Methods of treating disorders with Group I mGluR antagonists

ABSTRACT

mGluR5 antagonists are used for the treatment and prevention of disorders, including Fragile X, autism, mental retardation, schizophrenia and Down&#39;s Syndrome. The methods of the invention can be used to treat epilepsy and anxiety in a human having Fragile X syndrome, autism, mental retardation, schizophrenia and Down&#39;s Syndrome.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/114,433, filed Apr. 2, 2002, and a continuation-in-part ofInternational Application No. PCT/US02/10211, which designated theUnited States and was filed Apr. 2, 2002, published in English, both ofwhich claim the benefit of U.S. Application No. 60/280,915, filed Apr.2, 2001. The teachings of the above applications are incorporated hereinby reference in their entirety.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant NS39321from National Institute of Neurological Disease and Stroke. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

In the mammalian central nervous system (CNS), the transmission of nerveimpulses is controlled by the interaction between a neurotransmitterreleased by a sending neuron and a surface receptor on a receivingneuron, causing excitation of this receiving neuron. L-Glutamate, themost abundant neurotransmitter in the CNS, mediates the major excitatorypathway in mammals, and is referred to as an excitatory amino acid(EAA). The receptors that respond to glutamate are called excitatoryamino acid receptors (EAA receptors). See Watkins & Evans, AnnualReviews in Pharmacology and Toxicology, 21: 165 (1981), Monaghan,Bridges, and Cotman, Annual Reviews in Pharmacology and Toxicology, 29:365 (1989); Watkins, Krogsgaard-Larsen, and Honore, Transactions inPharmaceutical Science, 11: 25 (1990).

Excitatory amino acid receptors are classified into two general types.Receptors that are directly coupled to the opening of cation channels inthe cell membrane of the neurons are termed “ionotropic.” This type ofreceptor has been subdivided into at least three classes, which aredefined by the depolarizing actions of the selective agonistsN-methyl-D-aspartate (NMDA),α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainicacid (KA). Five kainate receptors, classified as either high affinity(KA1 and KA2) or low affinity (GluR5, GluR6 and GluR7) kainate receptorshave been identified. (Bleakman et al, Molecular Pharmacology, 1996,Vol. 49, No. 4, pp. 581-585).

The second general type of receptor is the G-protein or secondmessenger-linked “metabotropic” excitatory amino acid receptor. Thissecond type is a highly heterogeneous family of glutamate receptors thatare linked to multiple second messenger systems. Based on their aminoacid sequence homology, agonist pharmacology, and coupling totransduction mechanisms, the 8 presently known mGluR sub-types areclassified into three groups. Group I receptors (mGluR1 and mGluR5) havebeen shown to be coupled to stimulation of phospholipase C resulting inphosphoinositide hydrolysis and elevation of intracellular Ca⁺⁺ levels,and, in some expression systems, to modulation of ion channels, such asK⁺ channels, Ca⁺ channels, non-selective cation channels, or NMDAreceptors. Group II receptors (mGluR2 and mGluR3) and Group IIIreceptors (mGluRs4, 6, 7, and 8) are negatively coupled to adenylcyclaseand have been shown to couple to inhibition of cAMP formation whenheterologously expressed in mammalian cells, and to G-protein-activatedinward rectifying potassium channels in Xenopus oocytes and in unipolarbrush cells in the cerebellum. Besides mGluR6, which is essentially onlyexpressed in the retina, the mGluR5 are felt to be widely expressedthroughout the central nervous system.

Both types of receptors appear not only to mediate normal synaptictransmission along excitatory pathways, but also participate in themodification of synaptic connections during development and throughoutlife. Schoepp, Bockaert, and Sladeczek, Trends in PharmacologicalScience, 11: 508 (1990); McDonald and Johnson, Brain Research Reviews,15: 41 (1990).

The excessive or inappropriate stimulation of excitatory amino acidreceptors leads to neuronal cell damage or loss by way of a mechanismknown as excitotoxicity. This process has been suggested to mediateneuronal degeneration in a variety of conditions. Agonists andantagonists of these receptors may be useful for the treatment of acuteand chronic neurodegenerative conditions.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of treating or preventingdisorders, including but not limited to Fragile X syndrome, Down'sSyndrome, and other forms of mental retardation, schizophrenia, andautism, comprising administering to a patient (e.g., a human) anantagonist of Group I mGluR (mGluR1 or mGluR5). In a particularembodiment, the mGluR antagonist is selective for mGluR5, e.g., in thehippocampus. The present invention provides:

-   a) the use of an mGluR antagonist for the treatment of Down's    Syndrome, Fragile X and other forms of mental retardation,    schizophrenia and autism,-   b) the use of an mGluR antagonist in the manufacture of a    pharmaceutical composition for the treatment of Down's Syndrome,    Fragile X and other forms of mental retardation, schizophrenia and    autism,-   c) a method of treating Down's Syndrome, Fragile X and autism in a    subject in need of such treatment, comprising administration to such    subject of a therapeutically effective amount of an mGluR    antagonist, and-   d) a method of treating Down's Syndrome, Fragile X and other forms    of mental retardation, schizophrenia and autism in a subject in need    of such treatment, comprising administration to such subject of a    therapeutically effective amount of a pharmaceutical composition    comprising an mGluR antagonist.

Certain embodiments of the invention relate to a method for treatingDown's Syndrome, Fragile X and other forms of mental retardation,schizophrenia and autism, comprising co-administering other therapeuticagents (e.g., simultaneously or at different times) to the patient(human or other animal) with an amount of an mGluR antagonist sufficientto treat the disorder. In certain embodiments, the composition is fororal administration or for transdermal administration.

In another aspect of the invention, the mGluR antagonist is a selectivemGluR5 antagonist.

In another aspect of the invention, the mGluR antagonist is selectedfrom 6-methyl-2-(phenylazo)-3-pyridinol, α-methyl-4-carboxyphenylglycine(MCPG), 2-methyl-6-(phenylethynyl)-pyridine (MPEP),3S,4aR,6S,8aRS-6-((((1H-tetrazole-5-yl)methyl)oxy)methyl)-1,2,3,4,4a,5,6,7,8,8α-decahydroisoquinoline-3-carboxylicacid, 2-methyl-6-[(1E)-2-phenylethynyl]-pyridine,(E)-6-methyl-2-styryl-pyridine (SIB 1893), LY293 558,6-(((4-carboxy)phenyl)methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylicacid,3S,4aR,6S,8aR-6-((((1H-letrazole-5-yl)methyl)oxy)methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylicacid, and3S,4aR,6S,8aR-6-(((4-carboxy)-phenyl)methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylicacid, and their pharmaceutically acceptable salts, analogues andderivatives thereof. In other aspects, the mGluR5 receptor antagonist isformulated with a pharmaceutically acceptable diluent or carrier.

Another aspect of the invention is a kit comprising one or more mGluRantagonists, provided in single oral dosage form or as a transdermalpatch, in an amount sufficient for treating neurological disordersselected from Fragile X, Down's Syndrome, and other forms of mentalretardation, autism, and schizophrenia in a patient, and in associationwith instructions (written and/or pictorial) describing the use of thekit for treating neurological disorders, and optionally, warnings ofpossible side effects and drug—drug or drug-food interactions.

One aspect of the present invention is a method for conducting apharmaceutical business. Accordingly, one embodiment of the presentinvention is a method for conducting a pharmaceutical business,comprising:

-   -   a. manufacturing a kit comprising one or more mGluR antagonists,        provided in single oral dosage form or as a transdermal patch,        in an amount sufficient for treating neurological disorders        selected from Fragile X, Down's Syndrome and other forms of        mental retardation, autism, and schizophrenia in a patient, and        in association with instructions (written and/or pictorial)        describing the use of the kit for treating neurological        disorders, and optionally, warnings of possible side effects and        drug—drug or drug-food interactions; and    -   b. marketing to healthcare providers the benefits of using the        kit to treat neurological disorders of patients.

Another embodiment of the present invention is a method for conducting apharmaceutical business, comprising:

-   -   a. providing a distribution network for selling a kit comprising        one or more mGluR5 antagonists, provided in single oral dosage        form or as a transdermal patch, in an amount sufficient for        treating neurological disorders selected from Fragile X, Down's        Syndrome and other forms of mental retardation, autism, and        schizophrenia in a patient, and in association with instructions        (written and/or pictorial) describing the use of the kit for        treating neurological disorders, and optionally, warnings of        possible side effects and drug—drug or drug-food interactions;        and    -   b. providing instruction material to patients or physicians for        using the kit to treat neurological disorders of patients.

Another embodiment of the present invention is a method for conducting apharmaceutical business, comprising:

-   -   a. determining an appropriate dosage of an mGluR antagonist to        treat neurological disorders in a class of patients;    -   b. conducting therapeutic profiling of one or more formulations        of the mGluR5 antagonist identified in step (a), for efficacy        and toxicity in animals; and    -   c. providing a distribution network for selling a the        formulations identified in step (b) as having an acceptable        therapeutic profile.

In certain embodiments, the invention provides a method which includesan additional step of providing a sales group for marketing thepreparation to healthcare providers.

Further still, the present invention discloses a method for conducting apharmaceutical business, comprising:

-   -   a. determining an appropriate dosage of an mGluR antagonist to        treat a neurological disorder in a class of patients; and    -   b. licensing, to a third party, the rights for further        development and sale of the mGluR5 antagonist for treating the        neurological disorder.

In yet another aspect, the invention relates to a method for preparing apharmaceutical preparation, comprising combining an mGluR antagonist anda pharmaceutically acceptable explicit in a composition for simultaneousadministration of the drug.

In still another aspect, the invention relates to a method forconducting a pharmaceutical business, by manufacturing a preparation ofan mGluR antagonist (or prodrug or metabolite thereof) or a kitincluding separate formulations of each, and marketing to healthcareproviders the benefits of using the preparation or kit in the treatmentof Down's Syndrome, Fragile X and other forms of mental retardation,autism, and schizophrenia.

In yet another aspect, the invention provides a method for conducting apharmaceutical business, by providing a distribution network for sellingthe combinatorial preparations and kits, and providing instructionmaterial to patients or physicians for using such preparation to treatDown's Syndrome, Fragile X and other forms of mental retardation,autism, and schizophrenia.

In still a further aspect, the invention relates to a method forconducting a pharmaceutical business, by determining an appropriateformulation and dosage of an mGluR antagonist. In certain embodiments,the method further includes an additional step of providing a salesgroup for marketing the preparation to healthcare providers.

In yet another aspect, the invention provides a method for conducting apharmaceutical business by determining an appropriate formulation anddosage of an mGluR antagonist, and licensing, to a third party, therights for further development and sale of the formulation. In anotheraspect, the class of patients suffers from neurological disorders.

In other embodiments, the method comprises administering to the patientan effective amount of the in mGluR antagonist or combinations thereof.In another embodiment, the mGluR antagonist is administered in a doseranging from about 10 to about 1000 mg/kg body weight/day. In oneembodiment, the mGluR antagonist is administered in a dose ranging fromabout 50 to about 800 mg/kg body weight/day. In another embodiment, themGluR antagonist is administered in a dose ranging from about 250 toabout 500 mg/kg body weight/day.

In certain embodiments, the mGluR antagonist has an ED₅₀ of 10 μM, 1 μM,100 nm, 10 nm, or less. In one embodiment, the TI is 10, 100, 1000, orgreater. In certain embodiments, the ED₅₀ for group I receptorantagonism is at least 10 times less than the ED₅₀ for each of group IIor group III receptor antagonism, e.g., mGluR2, mGluR3, mGluR4, mGluR6,mGluR7, and mGluR8. The methods of the invention can be used to treatneurological conditions (e.g., Fragile X syndrome, mental retardation).

In yet another embodiment, the invention is directed to a method oftreating anxiety in a human having Fragile X syndrome, comprising thestep of administering to the human a Group I mGluR antagonist.

In still another embodiment, the invention is a method of treating anepilepsy in a human having Fragile X syndrome, comprising the step ofadministering to the human a Group I mGluR antagonist.

An additional embodiment of the invention is a method of treatinganxiety in a human having a disorder selected from the group consistingof autism, mental retardation and Down's Syndrome, comprising the stepof administering to the human a Group I mGluR antagonist.

In another embodiment, the invention is a method of treating an epilepsyin a human having a disorder selected from the group consisting ofautism, mental retardation and Down's Syndrome, comprising the step ofadministering to the human a Group I mGluR antagonist.

Treatment of humans with Group I mGluR antagonists can halt, diminish,inhibit, reverse or ameliorate conditions associated with mentalretardation (e.g., anxiety, epilepsy), thereby increasing the quality oflife for humans afflicted with mental retardation conditions.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 shows (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced long-termdepression (LTD).

FIG. 1A depicts the dose dependent effects of DHPG application (5 min;indicated by the downward arrow) on field potential (FP) slope values(10 μM DHPG; n=5; 50 μM; n=11; 100 μM DHPG; n=4). Inset: schematic ofplacement of stimulating (S) and extracellular recording (R) electrodesin an isolated CA1 hippocampal slice. Representative field potentials(2-min average) from a slice treated with 50 μM DHPG and taken at thetimes indicated by the numbers on the graph. Calibration: 0.5 mV, 5 ms.

FIG. 1B shows that DHPG-LTD is stimulation independent. Inset: placementof stimulating electrodes (S1 and S2) that stimulated 2 independentinputs in alternation. Stimulation to I pathway (OFF path; o) was turnedoff immediately prior to DHPG application and resumed 30 min after DHPGwash out, while the other (ON path; ●) input was stimulated at baselinefrequency (0.067 Hz) for the duration of the experiment. A similarmagnitude of depression was observed in both the ON and OFF paths (n=4).

FIG. 1C shows that DHPG-LTD is saturable. Two applications of DHPG aresufficient to saturate LTD. A 3rd DHPG application did not induce anyfurther depression (n=8).

FIG. 1D shows that DHPG (50 μM; 5 min) application induces a persistentdepression of average excitatory postsynaptic potential (EPSP) slopevalues (n=6). Representative EPSP waveforms (2-min average) taken froman experiment at times indicated by numbers on the graph. Calibration: 5mV, 10 ms.

FIG. 1E shows that DHPG (50 μM; 5 min) application decreases excitatorypostsynaptic current (EPSC) amplitudes. Cells were voltage clamped at−70 mV. Recording mode was switched from voltage clamp to current (I)clamp during and 5 min after DHPG application as indicated by the bar.Representative EPSCs (2-min average) taken from an experiment at thetimes indicated by the numbers on the graph. Calibration: 125 pA, 25 ms.

FIGS. 2A-2C show that DHPG-LTD, but not N-methyl-D-aspartate receptor(NMDAR)-dependent LTD, require mGluR5.

FIG. 2A shows that DHPG-LTD is NMDAR independent. Preincubation ofslices in D-2-amino-5-phosphonopentanoic acid (AP5; 50 μM; ●; n=5) doesnot affect the magnitude of DHPG-LTD as compared with interleavedcontrol slices (◯; n=4).

FIG. 2B shows that DHPG-LTD requires mGluR5. DHPG application to slicesfrom homozygote mGluR5 knockout mice (−/−; ◯; n=8) does not induce LTD.Intermediate LTD is observed in heterozygotes (+/−; ▪; n=6) as comparedwith slices from wild-type mice (wt; ●; i=9).

FIG. 2C shows that low-frequency synaptic stimulation (LFS)-induced LTDdoes not require mGluR5. LFS induces a similar magnitude of LTD in bothhomozygote knockout mice (−/−; o; n=6) as compared with wild-type mice(wt, ●; n=6).

FIGS. 3A-3D show that DHPG-LTD is occluded by mGluR-dependent LTDinduced with PP-LFS, but not NMDAR-dependent LTD.

FIG. 3A shows the results of experiments when repeated episodes of LFSwere delivered to saturate NMDAR-dependent LTD. DHPG (downward arrow)was then applied to the slice.

FIG. 3B shows renormalized FP slope values to the pre-DHPG baseline(n=8).

FIG. 3C shows the results of experiments when repeated episodes ofPP-LFS were delivered to saturate mGluR-dependent LTD. DHPG (downwardarrow) was then applied to the slice. The entire experiment wasperformed in 50 μ. M D-AP5 to prevent induction of NMDAR-dependent LTD.

FIG. 3D shows renormalized FP slope values to the pre-DHPG baseline(n=).

FIG. 4A shows representative images of a control neuron and a neuron 15minutes after mGluR stimulation labeled via acid stripimmunocytochemistry for internalized GluR1. Scale bar, 10 μm.

FIG. 4B shows that quantification revealed a 2.5-fold increase in thedensity of internalized puncta as early as 15 min, lasting at least 60min.

FIG. 4C shows that mGluR-stimulated endocytosis of GluR1 is blocked by aGroup I mGluR antagonist, LY344545.

FIG. 4D shows that inhibition of protein synthesis by cycloheximide (60μM) treatment decreases mGluR-stimulated endocytosis.

FIGS. 5A and 5B show representative images of a control neuron stainedwith an antibody directed against the synaptic marker synapsin I (FIG.5A) and an antibody against the N-terminus of GluR2 (FIG. 5B) Scale bar,10 μm.

FIGS. 5C and 5D show higher magnification images of the same cell as inFIG. 5A demonstrating the colocalization of synapsin (FIG. 5C) and GluR2(FIG. 5D) Scale bar, 5 μm.

FIGS. 5E and 5F depict a similar degree of colocalization was observedwith antibodies against synaptophysin (FIG. 5E) and the N-terminus ofGluR1 (FIG. 5F).

FIGS. 5G and 5H show that no change in synapsin puncta density wasdetected 1 h after DHPG (FIG. 5G) but there was a large decrease in thenumber of synaptic GluR2 puncta (FIG. 5H) Scale bar, 10 μm.

FIG. 5I show that 80.6±9.0% of synapsin puncta colocalized with GluR2 oncontrol neurons. However, 1 h following DHPG, only 40.8±11% of synapseshad surface staining for GluR2.

FIGS. 5J and 5K show that GluR1-positive synapses are reduced by DHPGtreatment and the stable expression of this change is inhibited bycycloheximide. Only 29.3±5.4% of synaptophysin-positive synapsesexpressed GluR1 puncta 15 min after DHPG compared to 72.5±4.7% incontrol cultures. This effect of DHPG was not affected by cycloheximide(FIG. 5J). In contrast, cycloheximide significantly inhibited the lossof GluR1 measured 60 min following DHPG (FIG. 5K).

FIG. 6A shows a representative blot of samples of total and biotinylatedsurface GluR1 from a control culture (lanes 1 and 2) and 60 minfollowing DHPG treatment (lanes 3 and 4).

FIG. 6B depicts densitometric quantification 60 min following DHPG.Surface GluR1 levels were reduced to 56.8±4.0% of control levels.

FIG. 7A shows representative mEPSC recordings from a cell before and onehour after DHPG application.

FIG. 7B depicts cumulative probability histograms for inter-eventinterval and amplitude for the cell depicted in FIG. 7A before DHPG andin a period beginning 45 min after DHPG application.

FIG. 7C depicts group-averaged mEPSC amplitude and inter-event intervalbefore, 15 min and 1 h following DHPG application.

FIGS. 8A and 8B show representative images of a control neuron stainedwith an antibody directed against the synaptic marker synapsin I (FIG.8A) and an antibody to the N-terminus of NR1 (FIG. 8B). Scale bar, 10μm.

FIGS. 8C and 8D depict higher magnification images of the same cell asin

FIG. 8A demonstrating the colocalization of synapsin (FIG. 8C) and NR1(FIG. 8D). Scale bar, 5 μm.

FIGS. 8E and 8F show that no change in synapsin puncta density wasdetected 1 h after DHPG (FIG. 8E) but there was a large decrease in thenumber of synaptic NR1 puncta (FIG. 8F). Scale bar, 10 m.

FIG. 8G shows that DHPG reduced the percent of synapses positive for NR160 min after treatment onset and that this effect was inhibited bycycloheximide.

FIG. 8H shows a representative blot of total and biotinylated surfaceNR1 in control (lanes 1 and 2) and 60 minutes following treatment withDHPG (lanes 3 and 4; reprobe of blot in FIG. 3A).

FIG. 8I shows that after sixty minutes of DHPG treatment, surface NR1levels were reduced to 32.3±8.2% of control levels. Cycloheximidereduced the loss of surface NMDARs to 79.1±14.5% of control levels.

FIGS. 9A, 9B and 9C shows that DHPG application attenuates synapticallyevoked NMDAR-mediated EPSCs and NMDA-evoked currents. DHPG-induceddepression of synaptically evoked NMDAR EPSCs. Arrows indicate onset of5 min DHPG application. R_(S), series resistance.

FIG. 9B shows a two-minute average of NMDA-evoked current amplitudesbefore and after application of 100 μM DHPG.

FIG. 9C shows a two-minute average of control NMDA-evoked currents.

FIGS 10A, 10B and 10C show that the synaptic induction of mGluR-LTD(paired-pulse low-frequency stimulation (PP-LFS)) is significantlyenhanced in the hippocampus of Fragile X mental retardation Fmr1-KO(knock out) mice compared with WT (wild type) controls.

FIGS. 11A, 11B and 11C show that the mGluR agonist DHPG (5 min; 100 μM)induces a greater LTD of synaptic responses in the hippocampus ofFmr1-KO mice as compared to wild-type (WT) littermate controls.

FIGS. 12A, 12B and 12C show that the synaptic induction ofNMDAR-dependent LTD is comparable in Fmr1-KO mice and WT controls.

FIG. 13 depicts a model of the relation between Group I mGluR (e.g.,mGluR5) and FMRP. AMPAR isα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention, either as steps of theinvention or as combinations of parts of the invention, will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown bway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

Evidence that Fragile X mental retardation protein (FMRP) is involved inactivity-dependent local synaptic protein synthesis has only recentlyemerged. The major excitatory neurotransmitter glutamate, via group I metabotropic glutamate receptors (mGluRs), stimulates protein synthesis indendrites. The Group I mGluRs are a subgroup of the G-protein coupledmGluR family, and are composed of two subtypes, mGluR1 and mGluR5.Subsequent work demonstrated that FMR1 in RNA is present in dendritesand FMRP is synthesized in response to mGluR activation ofsynaptoneurosomes (Weiler, et al., 1997). Because FMRP itself canregulate mRNA translation, the synthesis of FMRP at synapses in responseof mGluR activation may be a mechanism by which neuronal activity canregulate or control synthesis of other proteins important for synapticplasticity and development.

Although it was known that mGluR activation can stimulate proteinsynthesis, and specifically that of FMRP, the functional role of thismechanism was unknown until recently. Several studies have demonstratedthat activation of group I mGluR5 with either synaptic stimulation orthe selective agonist R,S-dihydroxyphenylglycine (DHPG) induceslong-term depression (LTD) of synaptic responses in area CA1 of the rathippocampus (Fitzjohn et al., 1999; Kemp and Bashir, 1999; Huber et al.,2000). LTD is dependent on mGluR5 and most importantly requires therapid and dendritic synthesis of new proteins (Huber et al., 2000). ThisLTD mechanism provides clues to the function of glutamate oractivity-induced stimulation of local dendritic protein synthesis.

It has been suggested that an LTD-like mechanism could be responsiblefor elimination or pruning of inappropriate synapses which are formedduring early periods of postnatal development (Colman, et al., 1997;Bear and Rittenhouse, 1999). Recent evidence supports this hypothesis.Treatment of hippocampal neuronal cultures with the group I mGluRagonist, DHPG, results in a long-term decrease in the surface expressionof AMPA-subtype glutamate receptors (AMPAR), the receptors responsiblefor synaptic transmission at excitatory synapses. Like LTD, thelong-term decrease in the AMPAR surface expression is dependent onprotein synthesis (Snyder, et al., 2000). Preliminary data also indicatea concomitant reduction in the number of presynaptic terminals afterDHPG treatment. Together, these results indicate that activation ofmGluR5 results in decreases in synaptic strength most likely mediated bya reduction or elimination in the number of excitatory synapses. Thissynapse elimination process may contribute to the formation ofappropriate synaptic connections during development as well as in thestorage of memories in the adult.

The present invention is based on the discovery that FMRP plays anintegral part in the LTD mechanism. As described in detail below, therole of FMRP in LTD was discovered using the FMR1 knockout mouse modelof Fragile X syndrome. Briefly, hippocampal brain slices were preparedfrom either knockout or wildtype littermates. LTD was induced witheither DHPG application or a synaptic stimulation protocol, termedpaired-pulse low frequency stimulation (PP-LFS). Surprisingly, asignificant enhancement of LTD was observed in the knockout mice in boththe DHPG and PP-LFS treated slices. These results suggest that FMRP maynormally function as an inhibitor of mGluR-dependent protein synthesisand, in the absence of FMRP, there is unregulated synthesis of theproteins required for LTD. One implication of these results is that anexcess of LTD or a synapse elimination mechanism in FMR1 knockout miceor Fragile X patients may perturb the normal synaptic developmentprocess and lead to abnormalities in dendritic spine structure andeventually to cognitive deficits. Alternatively or in addition, theenhancement of an LTD-like mechanism in the adult could result in theineffective storage of information in the brain which could alsocontribute to mental retardation.

The discovery of a neuronal mechanism associated with mental retardationprovides therapies to prevent or reverse the synaptic abnormalities andcognitive deficits associated with Fragile X syndrome, Downe's syndromeand other forms of mental retardation, autism, schizophrenia and otherdisorders involving down-regulation of FMRP levels or expression. Forexample, treatment could be the administration of antagonists of Group ImGluR5, such as mGluR5, during early postnatal development to attenuatethe abnormally enhanced LTD and restore the balance of synapticformation and elimination. Furthermore, treatment of adults withantagonists of Group I mGluR5, such as mGluR5s, may reduce learningdeficits in light of evidence that neurons retain their ability to formdendrites and modulate surface expression of receptors for some time.

The present invention relates to the use of antagonists of Group ImGluRs, such as antagonists of mGluR5 and mGluR1, for treating Down'sSyndrome, Fragile X and other forms of mental retardation, schizophreniaand autism. An mGluR antagonist is a substance which diminishes orabolishes the effect of a ligand (or agonist) that activates an mGluR.Thus, the antagonist may be, for example, a chemical antagonist, apharmacokinetic antagonist, an antagonist by receptor block, anon-competitive antagonist, or a physiological antagonist.

Antagonists may act the level of the ligand-receptor interactions, suchas by competitively or non-competitively (e.g., allosterically)inhibiting ligand binding. In other embodiments, the antagonist may actdownstream of the receptor, such as by inhibiting receptor interactionwith a G protein or downstream events associated with G proteinactivation such as stimulation of phospholipase C, elevation inintracellular calcium, the production of or levels of cAMP oradenylcyclase, stimulation and/or modulation of ion channels (e.g., K+,Ca++). The antagonists can alter, diminish, halt, inhibit or prevent theabove-referenced cellular signaling events.

A “pharmacokinetic antagonist” effectively reduces the concentration ofthe active drug at its site of action, e.g., by increasing the rate ofmetabolic degradation of the active ligand. Antagonism by receptor-blockinvolves two important mechanisms: 1) reversible competitive antagonismand 2) irreversible, or non-equilibrium, competitive antagonism.Reversible competitive antagonism occurs when the rate of dissociationof the antagonist molecule from the receptor is sufficiently high that,on addition of the ligand, the antagonist molecules binding thereceptors are effectively replaced by the ligand. Irreversible ornon-equilibrium competitive antagonism occurs when the antagonistdissociates very slowly or not at all from the receptor, with the resultthat no change in the antagonist occupancy takes place when the ligandis applied. Thus, the antagonism is insurmountable. As used herein, a“competitive antagonist” is a molecule which binds directly to thereceptor or ligand in a manner that sterically interferes with theinteraction of the ligand with the receptor.

Non-competitive antagonism describes a situation where the antagonistdoes not compete directly with ligand binding at the receptor, butinstead blocks a point in the signal transduction pathway subsequent toreceptor activation by the ligand. Physiological antagonism looselydescribes the interaction of two substances whose opposing actions inthe body tend to cancel each other out. An antagonist can also be asubstance that diminishes or abolishes expression of functional mGluR.Thus, an antagonist can be, for example, a substance that diminishes orabolishes: 1) the expression of the gene encoding mGluR5, 2) thetranslation of mGluR5 RNA, 3) the post-translational modification ofmGluR5 protein, or 4) the insertion of GluR5 into the cell membrane.

II. Definitions

An “effective amount” refers to the amount of a compound including anmGluR antagonist that is effective, upon single or multiple doseadministration to a patient, in treating the patient suffering from thenamed disorder.

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect.

The term “IC₅₀” means the concentration of a drug which inhibits anactivity or property by 50%, e.g., by reducing the frequency of acondition, such as cell death, by 50%, by reducing binding of acompetitor peptide to a protein by 50% or by reducing the level of anactivity by 50%.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

“Composition” indicates a combination of multiple substances into anaggregate mixture.

The term “prodrug” is intended to encompass compounds which, underphysiologic conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties, such as esters, which are hydrolyzed underphysiologic conditions to reveal the desired molecule. In otherembodiments, the prodrug is converted by an enzymatic activity of thehost animal.

The term “metabolites” refers to active derivatives produced uponintroduction of a compound into a biological milieu, such as a patient.

An “agonist” is a molecule which activates a certain type of receptor.For example, glutamate molecules act as agonists when they excite EMreceptors. By contrast, an “antagonist” is a molecule which prevents orreduces the effects exerted by an agonist on a receptor. The term“therapeutic index” refers to the therapeutic index (TI) of a drug,defined as LD₅₀/ED₅₀.

By “transdermal patch” is meant a system capable of delivery of a drugto a patient via the skin, or any suitable external surface, includingmucosal membranes, such as those found inside the mouth. Such deliverysystems generally comprise a flexible backing, an adhesive and a drugretaining matrix, the backing protecting the adhesive and matrix and theadhesive holding the whole on the skin of the patient. On contact withthe skin, the drug-retaining matrix delivers drug to the skin,permitting the drug to pass through the skin into the patient's system.

The term “statistically significant” as used herein means that theobtained results are not likely to be due to chance fluctuations at thespecified level of probability. The two most commonly specified levelsof significance are 0.05 (p=0.05) and 0.01 (p=0.01). The level ofsignificance equal to 0.05 and 0.01 means that the probability of erroris 5 out of 100 and 1 out of 100, respectively.

The term “healthcare providers” refers to individuals or organizationsthat provide healthcare services to a person, community, etc. Examplesof “healthcare providers” include doctors, hospitals, continuing careretirement communities, skilled nursing facilities, subacute carefacilities, clinics, multispecialty clinics, freestanding ambulatorycenters, home health agencies, and HMO's.

The term “distribution network” refers to individuals or organizationsthat are linked together and transfer goods from one individual,organization, or location to a plurality of other individuals,organizations, or locations.

The term “sales group” refers to an organization of individuals who areassociated with the selling of a certain product.

The term “licensing” refers to the granting of authority by the owner ofa patent or the holder of know-how to another, empowering the latter tomake or use the patented composition or method or the know-how.

III. Exemplary Compounds of the Invention.

A. Exemplary mGluR Antagonists

The present invention contemplates the use of Group I mGluR antagonists,preferably selective mGluR5 antagonists.

Exemplary mGluR5 antagonists include, without limitation,2-methyl-6-(phenylethynyl)-pyridine (MPEP),(E)-6-methyl-2-styryl-pyridine (SIB 1893), LY293558, 2-methyl-6-[(1E)-2-phenylethynyl]-pyridine, 6-methyl-2-(phenylazo)-3-pyridinol,(RS)-α-methyl-4-carboxyphenylglycine (MCPG),3S,4aR,6S,8aRS-6-((((1H-tetrazole-5-yl)methyl)oxy)methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylicacid,3S,4aR,6S,8aR-6-((((1H-tetrazole-5-yl)methyl)oxy)methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylicacid,3SR,4aRS,6SR,8aRS-6-(((4-carboxy)phenyl)methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylicacid and3S,4aR,6S,8aR-6-(((4-carboxy)-phenyl)methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid, and their pharmaceuticallyacceptable salts, analogues and derivatives thereof.

Antagonists of mGluR5 are also described in WO 01/66113, WO 01/32632, WO01/14390, WO 01/08705, WO 01/05963, WO 01/02367, WO 01/02342, WO01/02340, WO 00/20001, WO 00/73283, WO 00/69816, WO 00/63166, WO00/26199, WO 00/26198, EP-A-0807621, WO 99/54280, WO 99/44639, WO99/26927, WO 99/08678, WO 99/02497, WO 98/45270, WO 98/34907, WO97/48399, WO 97/48400, WO 97/48409, WO 98/53812, WO 96/15100, WO95/25110, WO 98/06724, WO 96/15099 WO 97/05109, WO 97/05137, U.S. Pat.No. 6,218,385, U.S. Pat. No. 5,672,592, U.S. Pat. No. 5,795,877, U.S.Pat. No. 5,863,536, U.S. Pat. No. 5,880,112, U.S. Pat. No. 5,902,817,allowed U.S. application Ser. Nos. 08/825,997, 08/833,628, 08/842,360,and 08/899,319, all of which are hereby incorporated by reference.

For example, different classes of mGluR5 antagonists are described in WO01/08705 (pp. 3-7), WO 99/44639 (pp. 3-11), and WO 98/34907 (pp. 3-20).

Another class of mGluR1 antagonists, antisense oligonucleotides, isdescribed in WO 01/05963. Antisense oligonucleotides to mGluR5 can beprepared by analogy and used to selectively antagonize mGluR5, asdesired.

Another class of mGluR5 antagonists is described in WO 01/02367 and WO98/45270. Such compounds generally have the formula:

-   -   wherein R represents H or a hydrolyzable hydrocarbon moiety such        as an alkyl, heteroalkyl, alkenyl, or aralkyl moiety.

In certain such embodiments, the isoquinoline system has thestereochemical array

(wherein, as is known in the art, a dark spot on a carbon indicateshydrogen coming out of the page, and a pair of dashes indicates ahydrogen extending below the plane of the page), the enantiomer thereof,of a racemic mixture of the two.

Another class of antagonists, described in WO 01/66113, has the formula:

wherein

-   -   R₁ denotes hydrogen, lower alkyl, hydroxyl-lower alkyl, lower        alkyl-amino, piperidino, carboxy, esterified carboxy, amidated        carboxy, unsubstituted or lower alkyl-, lower alkoxy-, halo-        and/or trifluoromethyl-substituted        N-lower-alkyl-N-phenylcarbamoyl, lower alkoxy, halo-lower alkyl        or halo-lower alkoxy;    -   R₂ denotes hydrogen, lower alkyl, carboxy, esterified carboxy,        amidated carboxy, hydroxyl-lower alkyl, hydroxyl, lower alkoxy        or lower alkanoyloxy,        4-(4-fluoro-benzoyl-piperidin-1-yl-carboxy,        4-t.butyloxycarbonyl-piperazin-1-yl-carboxy,        4-(4-azido-2-hydroxybenzoyl)-piperazin-1-yl-carboxy or        4-(4-azido-2-hydroxy-3-iodo-benzoyl)-piperazin-1-yl-carboxy;    -   R₃ represents hydrogen, lower alkyl, carboxy, lower        alkoxy-carbonyl, lower alkyl-carbamoyl, hydroxy-lower alkyl,        di-lower alkyl-aminomethyl, morpholinocarbonyl or        4-(4-fluoro-benzoyl)-piperazin-1-yl-carboxy;    -   R₄ represents hydrogen, lower alkyl, hydroxy, hydroxy-lower        alkyl, amino-lower alkyl, lower alkylamino-lower alkyl, di-lower        alkylamino-lower alkyl, unsubstituted or hydroxy-substituted        lower alkyleneamino-lower alkyl, lower alkoxy, lower        alkanoyloxy, amino-lower alkoxy, lower alkylamino-lower alkoxy,        di-lower alkylaino-lower alkoxy, phthalimido-lower alkoxy,        unsubstituted or hydroxy-or-2-oxo-imidazolidin-1-yl-substituted        lower alkyleneamino-lower alkoxy, carboxy, esterified or        amidated carboxy, carboxy-lower alkoxy or esterified        carboxy-lower alkoxy; and    -   X represents an optionally halo-substituted lower alkenylene or        alkynylene group bonded via vicinal saturated carbon atoms or an        azo (—N═N—) group, and R₅ denotes an aromatic or heteroaromatic        group which is unsubstituted or substituted by one or more        substituents selected from lower alkyl, halo, halo-lower alkyl,        halo-lower alkoxy, lower alkenyl, lower alkynyl, unsubstituted        or lower alkyl-, lower alkoxy-, halo- and/or        trifluoromethyl-substituted phenyl, unsubstituted or lower        alkyl-, lower alkoxy-, halo and/or trifluoromethyl-substituted        phenyl-lower alkynyl, hydroxy, hydroxy-lower alkyl, lower        alkanoyloxy-lower alkyl, lower alkoxy, lower alkenyloxy, lower        alkylenedioxy, lower alkanoyloxy, amino-, lower alkylamino-,        lower alkanoylamino- or N-lower alkyl-N-lower        alkanoylamino-lower alkoxy, unsubstituted or lower alkyl-, lower        alkoxy-, halo- and/or trifluoromethyl-substituted phenoxy,        unsubstituted or lower alkyl-, lower alkoxy-, halo and/or        trifluoromethyl-substituted phenyl-lower alkoxy, acyl, carboxy,        esterified carboxy, amidated carboxy, cyano, carboxy-lower        alkylamino, esterified carboxy-lower alkylamino, amidated        carboxy-lower alkylamino, phosphono-lower alkylamino-esterified        phosphono-lower alkylamino, nitro, amino, lower alkylamino,        di-lower alkylamino-acylamino, N-acyl-N-lower alkylamino,        phenylamino, phenyl-lower alkylamino, cycloalkyl-lower        alkylamino or heteroaryl-lower alkylamino each of which may be        unsubstituted or lower alkyl-, lower alkoxy-, halo- and/or        trifluoromethyl-substituted; their N-oxides and their        pharmaceutically acceptable salts.

In certain such embodiments, as disclosed in WO 01/66113 and WO00/20001, these compounds have the formula:

wherein

-   -   R₁ is hydrogen, (C₁₋₄)alkyl, (C₁₋₄)alkoxy, cyano, ethynyl or        di(C₁₋₄)alkylamino,    -   R₂ is hydrogen, hydroxy, carboxy, (C₁₋₄) alkoxycarbonyl,        di(C₁₋₄)alkylaminomethyl,        4-(4-fluoro-benzoyl)-piperidin-1-yl-carboxy,        4-t-butyloxycarbonyl-piperazin-1-yl-carboxy,        4-(4-azido-2-hydroxybenzoyl)-piperazin-1-yl-carboxy, or        4-(4-azido-2-hydroxy-3-iodo-benzoyl)-piperazin-1-yl-carboxy,    -   R₃ is hydrogen, (C₁₋₄)alkyl, carboxy, (C₁₋₄)alkoxycarbonyl,        (C₁₋₄)alkylcarbamoyl, hydroxy(C₁₋₄)alkyl,        di(C₁₋₄)alkylaminomethyl, morpholinocarbonyl or        4-(4-fluoro-benzoyl)-piperazin-1-yl-carboxy,    -   R₄ is hydrogen, hydroxyl, carboxy, C(₂₋₅)alkanoyloxy,        (C₁₋₄)alkoxycarbonyl, amino (C₁₋₄)alkoxy,        di(C₁₋₄)alkylamino(C₁₋₄)alkoxy, di(C₁₋₄)alkylamino(C₁₋₄)alkyl or        hydroxy(C₁₋₄)alkyl, and    -   R₅ is a group of formula        wherein    -   R_(a) and R_(b) independently are hydrogen, halogen, nitro,        cyano, (C₁₋₄)alkyl, (C₁₋₄)alkoxy, trifluoromethyl,        trifluoromethoxy or (C₂₋₅)alkynyl, and R_(c) is hydrogen,        fluorine, chlorine bromine, hydroxy-(C₁₋₄)alkyl,        (C₂₋₅)alkanoyloxy, (C₁₋₄)alkoxy, or cyano, and    -   R_(d) is hydrogen, halogen or (C₁₋₄)alkyl;    -   in free form or in the form of pharmaccutically acceptable        salts.        In certain other embodiments disclosed in WO 01/66113, mGluR5        antagonists have structures of the formula:        wherein R₆ is hydrogen, hydroxy, or C₁₋₆ alkoxy;    -   R₇ is hydrogen, carboxy, tetrazolyl, —SO₂H, —SO₃H, —OSO₃H,        —CONHOH, or —P(OH)OR′, —PO(OH)OR′, —OP(OH)OR′ or —OPO(OH)OR′        where R′ is hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, or aryl C₁₋₆        aryl;    -   R₈ is hydrogen, hydroxy or C₁₋₄ alkoxy; and    -   R₉ is fluoro, trifluoromethyl, nitro, C₁₋₆ alkyl, C₃₋₇        cycloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkylthio,        heteroaryl, optionally substituted aryl, optionally substituted        aryl C₁₋₆ alkyl, optionally substituted aryl C₂₋₆ alkenyl,        optionally substituted aryl C₂₋₆ alkynyl, optionally substituted        aryloxy, optionally substituted C₁₋₆ alkoxy, optionally        substituted arythio, optionally substituted aryl C₁₋₆ alkylthio,        —CONR″R′″, —NR″R′″, —OCONR″R′″ or —SONR″R′″, where R″ and R′″        are each hydrogen, C₁₋₆ alkyl or aryl C₁₋₆ alkyl, or R″ and R′″        together form a C₃₋₇ alkylene ring;    -   or a salt or ester thereof.

Yet another class of mGluR5 antagonists is described in WO 00/63166.These compounds have the formula:

wherein

-   -   R₁₀ signifies hydrogen or lower alkyl;    -   R₁₁ signifies, independently for each occurrence, hydrogen,        lower alkyl, lower alkoxy, halogen or trifluoromethyl;    -   X signifies O, S, or two hydrogen atoms not forming a bridge;    -   A¹/A² signify, independently from each other, phenyl or a        6-membered heterocycle containing 1 or 2 nitrogen atoms;    -   B is a group of formula        wherein    -   R¹² signifies lower alkyl, lower alkenyl, lower alkynyl, benzyl,        lower alkyl-cycloalkyl, lower alkyl-cyano, lower        alkyl-pyridinyl, lower alkyl-lower alkoxy-phenyl, lower        alkyl-phenyl (optionally substituted by lower alkoxy), phenyl        (optionally substituted by lower alkoxy), lower alkyl-thienyl,        cycloalkyl, lower alkyl-trifluoromethyl, or lower        alkyl-morpholinyl;    -   Y signifies —O—, —S— or bond;    -   Z signifies —O— or —S—;    -   or B is a 5-membered heterocyclic group of formulas        wherein

R¹³ and R¹⁴ independently signify hydrogen, lower alkyl, lower alkoxy,cyclohexyl, lower alkyl-cyclohexyl or trifluoromethyl, with the provisothat at least one of R¹³ or R¹⁴ is hydrogen;

as well as with their pharmaceutically acceptable salts.

Another class of mGluR1 antagonists is described in WO 01/32632. Thesecompounds have the formula:

-   X¹ represents O or NH;-   L represents a bond or a (1-6C) alkylene chain optionally    interrupted by O, S, SO, SO or NH and optionally substituted on an    alkylene carbon atom by fluoro, hydroxy, (1-4C)alkoxy or oxo;-   R¹ represents an unsubstituted or substituted carbocyclic or    heterocyclic group;-   R²represents a hydrogen atom, a halogen atom, a carboxyl group, a    cyano group, a SCH₂CN, or a group of formula X²-R⁵ in which X²    represents a bond, O, S, SO, SO₂ or NH and R⁵ represents    (1-8C)alkyl, (3-10C)cycloalkyl, halo(1-6C)alkyl, hydroxy(1-6C)alkyl,    dihydroxy(1-4C)alkyl, (1-4C)alkoxy(1-4C)alkyl,    (1-4C)alkanoyl(1-4C)alkyl, (1-4C)alkanoyloxy(1-4C)alkyl,    carboxy(1-4C)alkyl, (1-4C)alkylaminocarbonyl(1-4C)alkyl,    (1-4C)alkanoylamino, (1-4C)alkanoylamino(1-4C)alkyl,    (1-4C)alkanoylamino[(1-4C)alkyl]₂, (1-4C)alkylthio(1-4C)alkyl,    (1-4C)alkylsulfinyl(1-4C)alkyl, (1-4C)alkylsulfonyl(1-4C)alkyl,    (1-4C)alkylsulfonylamino)(1-4C)alkyl,    (1-4C)alkylamino-sulfonyl)(1-4C)alkyl,    di(1-4C)alkylaminophosphonyl)(1-4C)alkyl, phenyl or    phenyl(1-4C)alkyl in which any phenyl group is unsubstituted or    substituted by one or two substituents selected independently from a    halogen atom, (1-4C)alkyl and (1-4C)alkoxy; and-   R³ and R⁴ each independently represents (1-4C)alkyl or together with    the carbon atoms to which they are attached form an unsubstituted or    substituted carbocyclic or heterocyclic ring;    or a pharmaceutically acceptable salt thereof.

Another class of mGluR5 antagonists is described in WO 01/14390. Thesecompounds have the formula:

wherein,

-   either J and K are taken together with one or more additional atoms    independently selected from the group consisting of C, O, S, and N    in chemically reasonable substitution patterns to form a 3-7    membered saturated or unsaturated heterocyclic or carbocyclic ring,    and L is —CH,-   or J, K, and L are taken together with one or more additional atoms    independently selected from the group consisting of C, O, S, and N    in chemically reasonable substitution patterns to form a 4-8    membered saturated or unsaturated, mono-, bi-, or tricyclic, hetero-    or carbocyclic ring structure;-   Z is a metal chelating group;-   R₁ and R₂ are independently hydrogen, C₁-C₉ alkyl, C₂-C₉ alkenyl,    C₃-C₈ cycloalkyl, C₅-C₇ cycloalkenyl, or Ar, wherein each said    alkyl, alkenyl, cycloalkyl, cycloalkenyl, or Ar is independently    unsubstituted or substituted with one or more substituent(s); and-   Ar is a carbocyclic or heterocyclic moiety which is unsubstituted or    substituted with one or more substituent(s);    or a pharmaceutically acceptable equivalent thereof.

Still another class of mGluR5 antagonists is described in U.S. Pat. No.6,218,385. These compounds have the formula:

-   R¹ signifies hydrogen, hydroxy, lower alkyl, oxygen, halogen, or-   —OR, —O(C₃-C₆)cycloalkyl, —O(CHR)_(n)—(C₃-C₆)cycloalkyl, —O(CHR)_(n)    CN, —O(CHR)_(n) CF₃, —O(CHR)(CHR)_(n) NR2, —O(CHR)(CHR)_(n)OR,    —O(CHR)_(n)-lower alkenyl, —OCF₃, —OCF₂—R, —OCF₂-lower alkenyl,    —OCHRF, —OCHF-lower alkenyl, —OCF₂CRF₂, —OCF₂ Br, —O(CHR)_(n)CF₂Br,    —O(CHR)_(n)-phenyl, wherein the phenyl group may be optionally    substituted independently from each other by one to three lower    alkyl, lower alkoxy, halogen, nitro or cyano groups,-   —O(CHR)(CHR)_(n)-morpholino, —O(CHR)(CHR)_(n)-pyrrolidino,    —O(CHR)(CHR)_(n)-piperidino, —O(CHR) (CHR)_(n)-imidazolo,    —O(CHR)(CHR)_(n)-triazolo, —O(CHR)_(n)-pyridino,    —O(CHR)(CHR)_(n)—OSi-lower alkyl, —O(CHR)(CHR)_(n)OS(O)₂-lower    alkyl, —(CH₂)_(n)CH═CF₂, —O(CHR)_(n)-2,2-dimethyl-[1,3]dioxolane,    —O(CHR)_(n)—CHOR—CH₂OR, —O(CHR)_(n)—CHOR—(CHR)_(n)—CH₂OR or-   —SR or —S(CHR)_(n)COOR, or-   —NR2, —N(R)(CHR)(CHR)_(n)OR, —N(R)(CHR)_(n)CF₃,    —N(R)(CHR)(CHR)_(n)-1morpholino, —N(R)(CHR)(CHR)_(n)-imidazolo,    —N(R)(CHR)(CHR)_(n)-pyrrolidino,    —N(R)(CHR)(CHR)_(n)-pyrrolidin-2-one,    —N(R)(CHR)(CHR)_(n)-piperidino, —N(R)(CHR)(CHR)_(n)-triazolo,    —N(R)(CHR)_(n)-pyridino, or-   R¹ and R⁴ are interconnected to the groups—(CH₂)₃₋₅—, —(CH₂)₂—N═,    —CH═N—N=—, —CH═CH—N═, —NH—CH═CH— or-   —NR—CH₂—CH₂—and form together with any N or C atoms to which they    are attached an additional ring;-   n is 1-6,-   R signifies hydrogen, lower alkyl or lower alkenyl, independently    from each other, if more than one R is present;-   R₂ signifies nitro or cyano;-   R³ signifies hydrogen, lower alkyl, ═O, —S, —SR, —S(O)₂-lower alkyl,    —(C₃-C₆)cycloalky or piperazino, optionally substituted by lower    alkyl, or    -   —CONR₂, —(CHR)_(n)CONR₂, —(CHR)_(n)OR, —(CH₂)_(n)—CF₃, —CF₃,        —(CHR)_(n)OC(O)CF₃, —(CHR)_(n) COOR, —(CHR)_(n) SC₆H₅, wherein        the phenyl group may be optionally substituted independently        from each other by one to three lower alkyl, lower alkoxy,        halogen, nitro or cyano groups,    -   (CHR)_(n)— 1,3-dioxo-1,3-dihydro-isoindol,        —(CHR)_(n)-tetrahydro-pyran-2-yloxy or—(CHR)_(n)—S-lower alkyl,        or    -   NR₂, —NRCO-lower alkyl, —NRCHO, —N(R)(CHR)_(n)CN,        —N(R)(CHR)_(n)CF₃, —N(R)(CHR)(CHR)_(n)—OR,        —N(R)C(O)(CHR)_(n)O-lower alkyl, —NR(CHR)_(n)-lower alkyl,        —NR(CHR)(CHR)_(n)—OR, —N(R)(CHR)(CHR)_(n)—O-phenyl, wherein the        phenyl group may be optionally substituted independently from        each other by one to three lower alkyl, lower alkoxy, halogen,        nitro or cyano groups,    -   —N(R)(CHR)_(n)-lower alkenyl, —N(R)(CHR)(CHR)_(n),        —O—(CHR)_(r)OR, —N(R)(CHR)_(n)C(O)O-lower alkyl, —N(R)(CHR)_(n),        C(O)NR-lower alkyl, —N(R)(CH₂)_(n)-2,2-dimethyl-[1,3]dioxolane,        —N(R)(CHR)(CHR)_(n), morpholino, —N(R)(CHR)_(n)-pyridino,        —N(R)(CHR)(CHR)_(n)-piperidino, —N(R)(CHR)(CHR)_(n)-pyrrolidino,        —N(R)(CHR)(CHR), —O-pyridino, —N(R)(CHR)(CHR)_(n), imidazolo,        —N(R)(CHR)_(n)—CR₂—(CHR)_(n), —OR, —N(R)(CHR)_(n), —CR₂—OR,        —N(R)(CHR)_(n), —CHOR—CH₂OR,        —N(R)(CHR)_(n)—CHOR—(CHR)_(n)—CH₂OR, or    -   —OR, —O(CHR)_(n)CF₃, —OCF₃, —O(CHR)(CHR),_(n), —O-phenyl,        wherein the phenyl group maybe optionally substituted        independently from each other by one to three lower alkyl, lower        alkoxy, halogen, nitro or cyano groups,    -   —O(CHR)(CHR)_(n)—O-lower alkyl, —O(CHR)_(n)-pyridino or    -   —O(CHR)(CHR)_(n)-morpholino;    -   or R³ and R⁴ are interconnected to the groups—(CH₂)₃₋₅—,        —(CH₂)₂—N═, —CH═N—N=—, —CH═CH—N═, —NH—CH═CH—or    -   NR—CH₂—CH₂— and form together with any N or C atoms to which        they are attached an additional ring; and-   R⁴ signifies hydrogen, lower alkyl, lower alkenyl or nitro, or    -   —OR, —OCF₃, —OCF₂—R, —OCF₂-lower alkenyl, —OCHRF, —OCHF-lower        alkenyl, —O(CHR)_(n)CF₃, or    -   —(CHR)_(n)CHRF, —(CHR)_(n)CF₂ R, —(CHR)_(n)CF₃,        —(C₃-C₆)cycloalkyl, —(CHR)_(n)(C₃-C₆)cycloalkyl, —(CHR)_(n)CN,        —(CHR)_(n)-phenyl, wherein the phenyl group may be optionally        substituted independently from each other by one, to three lower        alkyl, lower alkoxy, halogen, nitro or cyano groups,    -   —(CHR)(CHR)_(n)OR, —(CHR)_(n)CHORCH₂OR; —(CHR)(CHR)_(n)NR₂,        —(CHR)_(n)COOR, —(CHR)(CHR)_(n)OSi-lower alkyl, —(CHR)(CHR)_(n),        —OS(O)₂-lower alkyl, —(CH₂)_(r)—CH═CF₂, —CF₃, —CF₂—R, —CF₂-lower        alkenyl, —CHRF, —CHF-lower alkenyl,        —(CHR)_(n)-2,2-dimethyl-[1,3]dioxolane,        —(CH₂)_(n)-2-oxo-azepan-1-yl, —(CHR)(CHR)_(n)-morpholino,        —(CHR)_(n)-pyridino, —(CHR)(CHR)_(n)-imidazolo,        —(CHR)(CHR)_(n)-triazolo, —(CHR)(CHR)_(n)-pyrrolidino,        optionally substituted by —(CH₂)_(n), OH,        —(CHR)(CHR)_(n)-3-hydroxy-pyrrolidino or        —(CHR)(CHR)_(n)-piperidino, or    -   —NR₂, —N(R)(CHR)_(n)-pyridino, —N(R)C(O)O-lower alkyl,        —N(CH₂CF₃)C(O)O-lower alkyl, —N[C(O)O-lower alkyl]₂,        —NR—NR—C(O)O-lower alkyl or —N(R)(CHR)_(n)CF₃, —NRCF₃, NRCF₂—R,        —NRCF₂-lower alkenyl, —NRCHRF, —NRCHP-lower alkenyl;    -   or is absent if X is —N=or ═N—;-   R⁵, R⁶ signify hydrogen, lower alkyl, lower alkoxy, amino, nitro,    —SO₂NH₂ or halogen; or-   R⁵ and R⁶ are interconnected to the group —O—CH₂—O— and form    together with the C atoms to which they are attached an additional    5-membered ring;-   R⁷, R⁹ signify hydrogen, lower alkyl, lower alkoxy, amino, nitro or    halogen;-   R⁹, R¹⁰ signify hydrogen or lower alkyl;-   R¹¹, R¹² signifies hydrogen, lower alkyl, hydroxy, lower alkoxy,    lower alkoxycarbonyloxy or lower alkanoyloxy;-   R¹³, R¹⁴ signify hydrogen, tritium or lower alkyl;-   R¹⁵, R¹⁶ signifies hydrogen, tritium, lower alkyl, hydroxy, lower    alkoxy or are together an oxo group; or-   X signifies—N═, ═N—, —N<, >C═ or ═C<;-   Y signifies—N═, ═N—, —NH—, —CH=or ═CH—; and-   the dotted line may be a bond when R¹, R or R⁴ represent a bivalent    atom, as well as with the pharmaceutically acceptable salts of each    compound of the above formula and the racemic and optically active    forms of each compound of the above formula.

Yet other classes of mGluR5 antagonists are described in WO 01/02342 andWO 01/02340. These compounds have the formulas, respectively:

stereoisomers thereof, or pharmaccutically acceptable salts or hydratesthereof, wherein: R1, and R2 are selected from the group comprising:

-   -   1) H; or    -   2) an acidic group selected from the group comprising carboxy,        phosphono, phosphino, sulfono, suloino, borono, tetrazol,        isoxazol, —(CH₂)_(n)-carboxy, —(CH₂)_(n)-phosphono,        —(CH₂)_(n)-phosphino, —(CH₂)_(n)-sulfono, —(CH₂)_(n)-sulfino,        —(CH₂)_(n)-borono, —(CH₂)_(n)-tetrazol, and —(CH₂)_(n)-isoxazol,        where n=1, 2, 3, 4, 5, or 6; or

-   X is an acidic group selected from the group comprising carboxy,    phosphono, phosphino, sulfono, sulfino, borono, tetrazol, isoxazol;

-   Y is a basic group selected from the group comprising 1° amino, 2°    amino, 3° amino, quaternary ammonium salts, aliphatic 1° amino,    aliphatic 2° amino, aliphatic 3° amino, aliphatic quaternary    ammonium salts, aromatic 1° amino, aromatic 2° amino, aromatic 3°    amino, aromatic quaternary ammonium salts, imidazol, guanidino,    boronoamino, allyl, urea, thiourea;

-   m is 0, 1;

-   R3, R4, R5, R6 are independently H, nitro, amino, halogen, tritium,    trifluoromethyl, trifluoroacetyl, sulfo, carboxy, carbamoyl,    sulfamoyl or acceptable esters thereof;    or a salt thereof with a pharmaceutically acceptable acid or base

Further classes of mGluR5 antagonists are described in WO 00/73283 andWO 99/26927. These compounds have the formula: R-[Linker]-Ar;

-   wherein R is an optionally substituted straight or branched chain    alkyl, arylalkyl, cycloalkyl, or alkylcycloalkyl group preferably    containing 5-12 carbon atoms. Ar is an optionally substituted    aromatic, heteroaromatic, arylalkyl, or heteroaralkyl moiety    containing up to 10 carbon atoms and up to 4 heteroatoms, and    [linker] is —(CH₂)_(n)-, where n is 2-6, and wherein up to 4 CH₂    groups may independently be substituted with groups selected from    the group consisting of C₁-C₃ alkyl, CHOH, CO, O, S, SO, SO₂, N, NH,    and NO. Two heteroatoms in the [linker] may not be adjacent except    when those atoms are both N (as in —N═N— of —NH—NH—) or are N and S    as in a sulfonamide. Two adjacent CH₂ groups in [linker] also may be    replaced by a substituted or unsubstituted alkene or alkyne group.    Pharmaceutically acceptable salts of the compounds also are    provided.

Another class of mGluR5 antagonists is described in WO 00/69816. Thesecompounds have the formula:

wherein,

-   n is O, 1 or 2;-   X is O, S, NH, or NOH;-   R¹ and R² are each independently H, CN, COOR, CONHR, C₁-C₆ alkyl,    tetrazole, or R and R² together represent “═O”;-   R is H or C₁-C₆ alkyl;    -   R³ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkyl, —CH₂OH,        —CH₂O-alkyl, —COOH;-   Ar is an unsubstituted or substituted aromatic or heteroaromatic    group;-   Z represents a group of the formulae    wherein,-   R⁴ and R⁵ are each independently H, halogen, C₁-C₆ alkoxy, —OAr,    C₁-C₆ alkyl, CF₃, COOR, CONHR, —CN, —OH, —COR, —S—(C₁-C₆ alkyl),    —SO₂(C₁-C₆ alkyl);-   A is CH₂, O, NH, NR, S, SO, SO₂, CH₂—CH₂, CH₂O, CHOH, C(O); wherein    R is as defined above;-   B is CHR, CR₂, C₁-C₆ alkyl, C(O), —CHOH, —CH₂—O, —CH═CH, CH₂—C(O),    CH₂—S, CH₂—S(O), CH₂—SO₂; —CHCO₂R; or —CH—NR₂, wherein R is as    defined above;-   Yet is a heterocycle such as furan, thiophene, or pyridine;    or a pharmaceutically acceptable salt thereof.

Yet other classes of mGluR1 antagonists are described in WO 00/26199 andWO 00/26198. These compounds have the formula:

in which,

-   R¹, R² and R³ are independently hydrogen, (C₁-C₆)alkyl,    (C₂-C₆)alkenyl, (C₃-C₁₀)cycloalkyl, unsubstituted or substituted    aryl, unsubstituted or substituted aryl(C₁-C₆)alkyl, unsubstituted    or substituted aryl(C₂-C₆)alkenyl, halo, carboxy,    (C₁-C₆)alkoxycarbonyl or —(CH₂)_(m)—OH, wherein m is 1, 2 or 3;-   --- indicates a single or a double bond;-   X and Y are each independently hydrogen, or X and Y together    represent a bridge of the formula —(CH₂)_(n)—,where n is 1 or 2;-   A₁ and A₂ are each independently an unsubstituted or substituted    aryl;-   Z is —CO—, —SO₂— or —CH2-; provided that, when Z is —CO—, A₁ is not    3,4,5-trimethoxyphenyl;    or a pharmaceutically acceptable salt or ester thereof.

Another class of mGluR5 antagonists is described in WO 99/54280. Thesecompounds have the formula:

wherein,

-   R1 can be an acidic group selected from the group consisting of    carboxyl, phosphono, phosphino, sulfono, sulfino, borono, tetrazol,    isoxazol, —CH₂-carboxyl, —CH₂-phosphono, —CH₂-phosphino,    —CH₂-sulfono, —CH₂-sulfino, —CH₂-borono, —CH₂-tetrazol,    —CH₂-isoxazol and higher homologues thereof,-   R2 can be a basic group selected from the group consisting of 1°    amino, 2° amino, 3° amino, quaternary ammonium salts, aliphatic 1°    amino, aliphatic 2° amino, aliphatic 3° amino, aliphatic quaternary    ammonium salts, aromatic 1° amino, aromatic 2° amino, aromatic 3°    amino, aromatic quaternary ammonium salts, imidazol, guanidino,    boronoamino, allyl, urea, thiourea;-   R3 can be H, aliphatic, aromatic or heterocyclic;-   R4 can be an acidic group selected from the group consisting of    carboxyl, phosphono, phosphino, sulfono, sulfino, borono, tetrazol,    isoxazol;-   stereoisomers thereof;    and pharmaceutically acceptable salts thereof.

Yet another class of mGluR5 antagonists is described in WO 99/08678.These compounds have the formula:

wherein R signifies halogen or lower alkyl;

-   n signifies 0-3;-   R¹ signifies lower alkyl; cycloalkyl; benzyl optionally substituted    by hydroxy, halogen, lower alkoxy or lower alkyl; benzoyl optionally    substituted by amino, lower alkylamino or di-lower alkylamino;    acetyl or cycloalkyl-carbonyl; and-   signifies an aromatic 5-membered residue which is bonded via a    N-atom and which contains further 1-3 N atoms in addition to the    linking N atom,    as well as their pharmaceutically acceptable salts.

Preferred antagonists are those that provide a reduction of activationby the ligand of at least 10%, and more preferably at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or even at least 99% at aconcentration of the antagonist, for example, of 1 μ g/ml, 10 μg/ml, 100μg/ml, 500 μg/ml, 1 mg/ml, 10 mg/ml, or 100 mg/ml. The percentageantagonism represents the percentage decrease in activity of mGluR,e.g., mGluR5, in a comparison of assays in the presence and absence ofthe antagonist. Any combination of the above mentioned degrees ofpercentage antagonism and concentration of antagonist may be used todefine an antagonist of the invention, with greater antagonism at lowerconcentrations being preferred.

An antagonist for use in the invention may be a relatively non-specificantagonist that is an antagonist of mGluRs in general. Preferably,however, an antagonist selectively antagonizes group I mGluR5. Even morepreferably, an antagonist used in the invention is a selectiveantagonist of mGluR5. A selective antagonist of mGluR5 is one thatantagonizes mGluR5, but antagonizes other mGluRs only weakly orsubstantially not at all, or at least antagonizes other mGluRs with anEC50 at least 10 or even 100 or 1000 times greater than the EC₅₀ atwhich it antagonizes mGluR5. Most preferred antagonists are those whichcan selectively antagonize mGluR5 at low concentrations, for example,those that cause a level of antagonism of 50% or greater at aconcentration of 100 μg/ml or less.

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g., a variegated library of potential mGluRantagonists, can be screened rapidly in high-throughput assays toidentify potential lead compounds, as well as to refine the specificity,toxicity, and/or cytotoxic-kinetic profile of a lead compound.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of compounds, such as chemically relatedcompounds, which may be screened together for a desired property. Thepreparation of many related compounds in a single reaction greatlyreduces and simplifies the number of screening processes which need tobe carried out. Screening for the appropriate physical properties can bedone by conventional methods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjectantagonists. See, for example, Blondelle et al. (1995) Trends Anal.Chem. 14: 83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899: theEllman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO94/08051; Chen et al. (1994) JACS 116: 2661: Kerr et al. (1993) JACS115: 252; PCT publications WO92/10092, WO93/09668 and WO91/07087; andthe Lerner et al. PCT publication WO93/20242). Accordingly, a variety oflibraries on the order of about 100 to 1,000,000 or more diversomers ofthe subject antagonists can be synthesized and screened for a particularactivity or property.

In an exemplary embodiment, a library of candidate antagonistdiversomers can be synthesized utilizing a scheme adapted to thetechniques described in the Still et al. PCT publication WO 94/08051,e.g., being linked to a polymer bead by a hydrolyzable or photolyzablegroup e.g., located at one of the positions of the candidate antagonistsor a substituent of a synthetic intermediate. According to the Still etal. technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead. The diversomers can be released from the bead, e.g., by hydrolysisand tested for activity.

A) Direct Characterization

A grouting trend in the field of combinatorial chemistry is to exploitthe sensitivity of techniques such as mass spectrometry (MS), forexample, which can be used to characterize sub-femtomolar amounts of acompound, and to directly determine the chemical constitution of acompound selected from a combinatorial library. For instance, where thelibrary is provided on an insoluble support matrix, discrete populationsof compounds can be first released from the support and characterized byMS. In other embodiments, as part of the MS sample preparationtechnique, such MS techniques as MALDI can be used to release a compoundfrom the matrix, particularly where a labile bond is used originally totether the compound to the matrix. For instance, a bead selected from alibrary can be irradiated in a MALDI step in order to release thediversomer from the matrix, and ionize the diversomer for MS analysis.

B) Multipin Synthesis

The libraries of the subject method can take the multipin libraryformat. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS 81:3998-4002) introduced a method for generating compound libraries by aparallel synthesis on polyacrylic acid-grated polyethylene pins arrayedin the microtitre plate format. The Geysen technique can be used tosynthesize and screen thousands of compounds per week using the multipinmethod, and the tethered compounds may be reused in many assays.Appropriate linker moieties can also been appended to the pins so thatthe compounds may be cleaved from the supports after synthesis forassessment of purity and further evaluation (c.f., Bray et al. (1990)Tetrahedron Lett 31: 5811-5814; Valerio et al. (1991) Anal Biochem 197:168-177; Bray et al. (1991) Tetrahedron Lett 32: 6163-6166).

C) Divide-Couple-Recombine

In yet another embodiment, a variegated library of compounds can beprovided on a set of beads utilizing the strategy ofdivide-couple-recombine (see, for example, Houghten (1985) PNAS 82:5131-5135; and U.S. Pat. Nos. 4,631,211; 5,440,016; 5,480,971). Briefly,as the name implies, at each synthesis step where degeneracy isintroduced into the library, the beads are divided into separate groupsequal to the number of different substituents to be added at aparticular position in the library, the different substituents coupledin separate reactions, and the beads recombined into one pool for thenext iteration.

In one embodiment, the divide-couple-recombine strategy can be carriedout using an analogous approach to the so-called “lea bag” method firstdeveloped by Houghten, where compound synthesis occurs on resin sealedinside porous polypropylene bags (Houghten et al. (1986) PNAS 82:5131-5135). Substituents are coupled to the compound-bearing resins byplacing the bags in appropriate reaction solutions, while all commonsteps such as resin washing and deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound.

D) Combinatorial Libraries by Light-Directed, Spatially AddressableParallel Chemical Synthesis

A scheme of combinatorial synthesis in which the identity of a compoundis given by its locations on a synthesis substrate is termed a spatiallyaddressable synthesis. In one embodiment, the combinatorial process iscarried out by controlling the addition of a chemical reagent tospecific locations on a solid support (Dower et al. (1991) Annu Rep MedChem 26: 271-280; Fodor, S.P.A. (1991) Science 251: 767; Pirrung et al.(1992) U.S. Pat. No. 5,143,854; Jacobs et al. (1994) Trends Biotechnol12: 19-26). The spatial resolution of photolithography affordsminiaturization. This technique can be carried out through the useprotection/deprotection reactions with photolabile protecting groups.

The key points of this technology are illustrated in Gallop et al.(1994)J Med Chem 37: 1233-1251. A synthesis substrate is prepared forcoupling through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers or otherphotolabile linkers. Light is used to selectively activate a specifiedregion of the synthesis support for coupling. Removal of the photolabileprotecting groups by light (deprotection) results in activation ofselected areas. After activation, the first of a set of amino acidanalogs, each bearing a photolabile protecting group on the aminoterminus, is exposed to the entire surface. Coupling only occurs inregions that were addressed by light in the preceding step. The reactionis stopped, the plates washed, and the substrate is again illuminatedthrough a second mask, activating a different region for reaction with asecond protected building block. The pattern of masks and the sequenceof reactants define the products and their locations. Since this processutilizes photolithography techniques, the number of compounds that canbe synthesized is limited only by the number of synthesis sites that canbe addressed with appropriate resolution. The position of each compoundis precisely known; hence, its interactions with other molecules can bedirectly assessed.

In a light-directed chemical synthesis, the products depend on thepattern of illumination and on the order of addition of reactants. Byvarying the lithographic patterns, many different sets of test compoundscan be synthesized simultaneously; this characteristic leads to thegeneration of many different masking strategies.

E) Encoded Combinatorial Libraries

In yet another embodiment, the subject method utilizes a compoundlibrary provided with an encoded tagging system. A recent improvement inthe identification of active compounds from combinatorial librariesemploys chemical indexing systems using tags that uniquely encode thereaction steps a given bead has undergone and, by inference, thestructure it carries. Conceptually, this approach mimics phage displaylibraries, where activity derives from expressed peptides, but thestructures of the active peptides are deduced from the correspondinggenomic DNA sequence. The first encoding of synthetic combinatoriallibraries employed DNA as the code. A variety of other forms of encodinghave been reported, including encoding with sequenceable bio-oligomers(e.g., oligonucleotides and peptides), and binary encoding withadditional non-sequenceable tags.

1) Tagging with Sequenceable Bio-Oligomers

The principle of using oligonucleotides to encode combinatorialsynthetic libraries was described in 1992 (Brenner et al. (1992) PNAS89: 5381-5383), and an example of such a library appeared the followingyear (Needles et at. (1993) PNAS 90: 10700-10704). A combinatoriallibrary of nominally 7⁷ (=823,543) peptides composed of all combinationsof Arg, Gin, Phe, Lys, Val, D-Val and Thr (three-letter amino acidcode), each of which was encoded by a specific dinucleotide (TA, TC, CT,AT, TT, CA and AC, respectively), was prepared by a series ofalternating rounds of peptide and oligonucleotide synthesis on solidsupport. In this work, the amine linking functionality on the bead wasspecifically differentiated toward peptide or oligonucleotide synthesisby simultaneously preincubating the beads with reagents that generateprotected OH groups for oligonucleotide synthesis and protected N112groups for peptide synthesis (here, in a ratio of 1:20). When complete,the tags each consisted of 69-mers, 14 units of which carried the code.The bead-bound library was incubated with a fluorescently labeledantibody, and beads containing bound antibody that fluoresced stronglywere harvested by fluorescence-activated cell sorting (FACS). The DNAtags were amplified by PCR and sequenced, and the predicted peptideswere synthesized. Following such techniques, compound libraries can bederived for use in the subject method, where the oligonucleotidesequence of the tag identifies the sequential combinatorial reactionsthat a particular bead underwent, and therefore provides the identity ofthe compound on the bead.

The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical lability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesisof non-oligomeric libraries. In preferred embodiments, the librariesemploy linkers permitting selective detachment of the test compoundlibrary member for assay.

Peptides have also been employed as tagging molecules for combinatoriallibraries. Two exemplary approaches are described in the art, both ofwhich employ branched linkers to solid phase upon which coding andligand strands are alternately elaborated. In the first approach (Kerret al. (1993) JACS 115: 2529-2531), orthogonality in synthesis isachieved by employing acid-labile protection for the coding strand andbase-labile protection for the compound strand.

In an alternative approach (Nikolaiev et at. (1993) Pept Res 6:161-170), branched linkers are employed so that the coding unit and thetest compound can both be attached to the same functional group on theresin. In one embodiment, a cleavable linker can be placed between thebranch point and the bead so that cleavage releases a moleculecontaining both code and the compound (Ptek et al. (1991) TetrahedronLett 32: 3891-3894). In another embodiment, the cleavable linker can beplaced so that the test compound can be selectively separated from thebead, leaving the code behind. This last construct is particularlyvaluable because it permits screening of the test compound withoutpotential interference of the coding groups. Examples in the art ofindependent cleavage and sequencing of peptide library members and theircorresponding tags has confirmed that the tags can accurately predictthe peptide structure.

2) Non-sequenceable Tagging: Binary Encoding

An alternative form of encoding the test compound library employs a setof non-sequencable electrophoric tagging molecules that are used as abinary code (Ohlmeyer et al. (1993) PNAS 90: 10922-10926). Exemplarytags are haloaromatic alkyl ethers that are detectable as theirtrimethylsilyl ethers at less than femtomolar levels by electron capturegas chromatography (ECGC). Variations in the length of the alkyl chain,as well as the nature and position of the aromatic halide substituents,permit the synthesis of at least 40 such tags, which in principle canencode 2⁴⁰ (e.g., upwards of 10¹²) different molecules. In the originalreport (Ohlmeyer et al., supra) the tags were bound to about 1% of theavailable amine groups of a peptide library via a photocleavableo-nitrobenzyl linker. This approach is convenient when preparingcombinatorial libraries of peptide-like or other amine-containingmolecules. A more versatile system has, however, been developed thatpermits encoding of essentially any combinatorial library. Here, thecompound would be attached to the solid support via the photocleavablelinker and the tag is attached through a catechol ether linker viacarbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem59: 4723-4724). This orthogonal attachment strategy permits theselective detachment of library members for assay in solution andsubsequent decoding by ECGC after oxidative detachment of the tag sets.

Although several amide-linked libraries in the art employ binaryencoding with the electrophoric tags attached to amine groups, attachingthese tags directly to the bead matrix provides far greater versatilityin the structures that can be prepared in encoded combinatoriallibraries. Attached in this way, the tags and their linker are nearly asunreactive as the bead matrix itself. Two binary-encoded combinatoriallibraries have been reported where the electrophoric tags are attacheddirectly to the solid phase (Ohlmeyer et al. (1995) PNAS 92: 6027-6031)and provide guidance for generating the subject compound library. Bothlibraries were constructed using an orthogonal attachment strategy inwhich the library member was linked to the solid support by aphotolabile linker and the tags were attached through a linker cleavableonly by vigorous oxidation. Because the library members can berepetitively partially photocluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: first,multiple beads are placed in 96-well microtiter plates; second,compounds are partially detached and transferred to assay plates; third,a metal binding assay identifies the active wells; fourth, thecorresponding beads are rearrayed singly into new microtiter plates;fifth, single active compounds are identified; and sixth, the structuresare decoded.

B. Exemplary mGLuR5 Antagonist Assays

Methods for identifying mGluR antagonists which may be used in a methodof treatment of the human or animal body by therapy, in particular inthe treatment of Down's Syndrome, Fragile X and other forms of mentalretardation, schizophrenia and autism, are known in the art. Suchmethods essentially comprise determining whether a test agent is anmGluR5 antagonist and determining whether an antagonist so identifiedcan be used in the treatment of Down's Syndrome, Fragile X, and/orautism.

One example of an assay for determining the activity of a test compoundas an antagonist of mGluR5 comprises expressing mGluR5 in CHO cellswhich have been transformed with cDNAs encoding the mGluR5 receptorprotein (Daggett et al., 1995, Neuropharmacology, 34, 871). The mGluR5is then activated by the addition of quisqualate and/or glutamate andcan be assessed by, for example the measurement of: (1) phosphoinositolhydrolysis (Litschig et al., 1999, Mol. Pharmacol. 55, 453); (ii)accumulation of [3H] cytidinephosphate-diacylglycerol (Cavanni et al.,1999, Neuropharmacology 38, A10); or fluorescent detection of calciuminflux into cells Kawabata et al., 1996, Nature 383, 89-1; Nakahara etal., 1997, J. Neurochemistry 69, 1467). The assay may be carried outboth in the presence and absence of a test product in order to determinewhether the test compound can antagonize the activity of the testproduct. This assay is amenable to high throughput screening.

GluR5 receptor antagonists may also be identified by radiolabelledligand binding studies at the cloned and expressed human GluR5 receptor(Korczak et al., 1994, Recept. Channels 3; 41-49), by whole cell voltageclamp electro-physiological recordings of functional activity at thehuman GluR5 receptor (Korczak et al., 1994, Recept. Channels 3; 41-49)and by whole cell voltage clamp electro-physiological recordings ofcurrents in acutely isolated rat dorsal root ganglion neurons (Bleakmanet al., 1996, Mol. Pharmacol. 49; 581-585).

Suitable control experiments can be carried out. For example, a putativeantagonist of mGluR5 could be tested with mGluR1 in order to determinethe specificity of the putative antagonist, or other receptors unrelatedto mGluRs to discount the possibility that it is a general antagonist ofcell membrane receptors.

Suitable test products for identifying an mGluR5 antagonist includecombinatorial libraries, defined chemical identities, peptides andpeptide mimetics, oligonucleotides and natural product libraries. Thetest products may be used in an initial screen of, for example, tenproducts per reaction, and the products of batches that show antagonismtested individually. Furthermore, antibody products (for example,monoclonal and polyclonal antibodies, single chain antibodies, chimericbodies and CDR-grafted antibodies) may be used.

C. Pharmaceutical Preparations of the mGluR5 Antagonists

In another aspect, the present invention provides pharmaceuticalpreparations comprising the subject mGluR5 antagonists. The mGluR5antagonists for use in the subject method may be conveniently formulatedfor administration with a biologically acceptable, non-pyrogenic, and/orsterile medium, such as water, buffered saline, polyol (for example,glycerol, propylene glycol, liquid polyethylene glycol and the like) orsuitable mixtures thereof. The optimum concentration of the activeingredient(s) in the chosen medium can be determined empirically,according to procedures well known to medicinal chemists. As usedherein, “biologically acceptable medium” includes any and all solvents,dispersion media, and the like which may be appropriate for the desiredroute of administration of the pharmaceutical preparation. The use ofsuch media for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe activity of the mGluR5 antagonists, its use in the pharmaceuticalpreparation of the invention is contemplated. Suitable vehicles andtheir formulation inclusive of other proteins are described, forexample, in the book Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA1985). These vehicles include injectable “deposit formulations.”

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g., pharmaceutical preparations of the mGluR5antagonist suitable for veterinary uses, e.g., for the treatment oflivestock or domestic animals, e.g., dogs.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs. A variety of biocompatible polymers (including hydrogels),including both biodegradable and non-degradable polymers, can be used toform an implant for the sustained release of an mGluR5 antagonist at aparticular target site.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are, of course, given byforms suitable for the desired administration route. For example, theymay be administered in tablets or capsule form, by injection,inhalation, eye lotion, ointment, suppository, controlled release patch,etc. administration by injection, infusion or inhalation; topical bylotion or ointment; and rectal by suppositories. Oral and topicaladministrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracistemally and topically, as by powders, ointments ordrops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular reuptake inhibitors employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with other antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticeffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The pharmaceutical compositionaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, andpastes for application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, or intravenous injection as,for example, a sterile solution or suspension; (3) topical application,for example, as a cream, ointment or spray applied to the skin; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam. However, in certain embodiments the subject compounds may besimply dissolved or suspended in sterile water.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filter, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject regulatorsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol, (12) esterssuch as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present mGluR5 antagonistsmay contain a basic functional group, such as amino or alkylamino, andare, thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These sails can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge ct al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66: 1-19).

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar—agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar—agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active reuptake inhibitor.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the reuptake inhibitors inthe proper medium. Absorption enhancers can also be used to increase theflux of the reuptake inhibitors across the skin. The rate of such fluxcan be controlled by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”, W.H. Freedman and Co., San Francisco, U.S.A., 1969 or “Livestock Feeds andFeeding” O and B books, Corvallis, Ore., U.S.A., 1977).

V. Exemplary Uses of the Compounds of the Invention

In one embodiment, the invention is directed to the use of an antagonistof a Group I mGluR, such as mGluR5, preferably of human mGluR5, in themanufacture of a medicament for use in a method of treating andpreventing a mental condition such as Down's Syndrome, Fragile X, andother forms of mental retardation, schizophrenia or autism. In variousembodiments, the present invention contemplates modes of treatment andprophylaxis which utilize one or more of the subject mGluR antagonists.

In another embodiment, the invention is an antagonist of a Group ImGluR, such as mGluR5, for use in a method of treatment of the human oranimal body by therapy; a method of treating a host suffering fromFragile X, Down's Syndrome, and other forms of mental retardation,schizophrenia or autism, which method comprises administering to thehost a therapeutically effective amount of an antagonist of mGluR5; apharmaceutical composition comprising an antagonist of a Group I mGluR,such as mGluR5, and a pharmaceutically acceptable carrier or diluent; ora product containing an antagonist of a Group I mGluR, such as mGluR5,and a therapeutic substance as a combined preparation.

The neural mechanisms which underlie mental retardation are largelyunknown. The discovery of the genetic basis of some forms of mentalretardation, such as Fragile X syndrome, has provided insight into thecellular mechanisms responsible for the cognitive deficits associatedwith mental retardation. Fragile X syndrome is the most common inheritedform of mental retardation, affecting I in 1500 men and I in 2500 women(de Vries, et al., 1993). Many patients with Fragile X syndrome exhibitmany neurological deficiencies or conditions, including moderate tosevere mental retardation (IQ=30-70), seizures (e.g., benign childhoodepilepsy, temporal lobe epilepsy), visual spatial defects, anxiety,learning difficulties and certain characteristics of autism.

An embodiment of the invention is a method of treating a human withFragile X syndrome, autism, Down's Syndrome, a neurological disorder ormental retardation to diminish, halt, ameliorate or prevent one or moreof the above-mentioned deficiencies (e.g., anxiety, benign childhoodepilepsy). In another embodiment, the invention is a method of treatinga human without Fragile X syndrome who suffers from one or more of theabove-mentioned deficiencies or conditions (e.g., anxiety, epilepsy).

In a particular embodiment, children with mental retardation, autism,Down's Syndrome and Fragile X syndrome can be treated with Group I mGluRantagonists. The children can be treated during infancy (between about 0to about 1 year of life), childhood (the period of life between infancyand puberty) and during puberty (between about 8 years of life to about18 years of life). In still another embodiment, the methods of theinvention can be used to treat adults (greater than about 18 years oflife) having mental retardation, Fragile X syndrome, schizophrenia,autism and Down's Syndrome. In a further embodiment of the invention,anxiety and epilepsy in children and adults having Fragile X syndrome,autism, mental retardation, Down's Syndrome and schizophrenia can betreated by administering to the children or the adult a Group I mGluRantagonist. In a particular embodiment, the Group I mGluR antagonist isan antagonist of mGluR5.

Unlike other forms of mental retardation, Fragile X patients exhibit nogross neuroanatomical deformities thought to give rise to cognitivedeficits (Hinton, et al., 1991; Wisniewski, et al., 1991). Instead,there is a neuropathology on a smaller scale, at the level of thesynapse. Cortical neurons of patients with Fragile X syndrome arecharacterized by reduced dendritic length and a number of irregular,very long, thin and tortuous dendritic spines, and a reduction inmature, short and stubby spines. These long, thin spines resembleimmature spines or dendritic filopodia prevalent in developing neuronsduring synapse maturation (Fiala, et al., 1998). Similar dendriticpathologies are associated with other forms of mental retardation suchas Down's or Rett syndrome (Marin-Padilla, 1972; Kaufmann and Moser,2000). Therefore, malfunctions of dendritic development and function maybe a common mechanism that underlies mental retardation.

The molecular basis for Fragile X syndrome was discovered when it wasfound that Fragile X patients have an expansion in the 5′ untranslatedregion of the Fragile X mental retardation (FMR1) gene, which results intranscriptional silencing (reviewed by (Imbert, et al., 1998)).Therefore, the loss of the FMR1 gene product, Fragile X mentalretardation protein (FMRP), is responsible for the Fragile X phenotype(Pieretti, et al., 1991; Verheij, et al., 1993). In support of thishypothesis, a mouse model of Fragile X syndrome was developed by a‘knockout’ (KO) of the FMR1 gene (Bakker and Consortium, 1994). TheFMR1-KO mice have many of the symptoms of the human Fragile X syndromeincluding learning deficits and hyperactivity (Fisch, et al., 1999;Paradee, et al., 1999).

Studies of the normal function of FMRP have indicated that FMRP is aregulator of protein synthesis or mRNA translation. FMRP has 2 RNAbinding regions and associates with translating polyribosomes and asubset of brain mRNAs. (Khandjian et al., 1996; Tarnanini et al., 1996).A very rare but severe form of Fragile X syndrome is caused by a singleamino acid mutation (1304N) in one of the RNA binding domains of FMRP.The severity of the Fragile X phenotype observed with the 1304N mutationindicates that RNA binding and association with polyribosomes is crucialto the function of FMRP (Siomi, et al., 1994). Interestingly, it is nowknown that polyribosomes and FMRP are present in dendritic spines andsynapses have the ability to synthesize protein suggesting that FMRP mayfunction specifically to regulate protein synthesis locally at synapses(Steward and Reeves, 1988; Feng, et al., 1997).

In recent years, a number of studies have demonstrated that mechanismsof activity-dependent synaptic strengthening or weakening, such aslong-term potentiation (LTP) or long-term depression (LTD) respectively,contribute to synapse formation and maturation (reviewed by (Collin, etal., 1997; Constantine-Paton and Cline, 1998). Both LTP and LTD canreflect a change in the level of surface density of neurotransmitterreceptors in the synaptic region of neuron membranes. For example thesurface expression of either or both of the NMDA receptor or the AMPAreceptor may be reduced in LTD or increased in LTP. Therefore, analteration in activity-dependent synaptic plasticity during synapsematuration may be one underlying source of the spine abnormalities andFragile X phenotype. Furthermore, persistent modifications at the levelof the synapse are thought to be the neural basis of learning and memoryin the adult. Altered activity-dependent plasticity in mature brains ofaffected patients may be a factor in the learning deficienciesexperienced in Fragile X syndrome.

Autism is a disabling neurological disorder that affects thousands ofAmericans and encompasses a number of subtypes, with various putativecauses and few documented ameliorative treatments. The disorders of theautistic spectrum may be present at birth, or may have later onset, forexample, at age two or three. There are no clear-cut biological markersfor autism. Diagnosis of the disorder is made by considering the degreeto which the child matches the behavioral syndrome, which ischaracterized by deficits in sociability, reciprocal verbal andnonverbal communication along with restricted, repetitive orstereotypical behavior.

A genetic basis for autism is suggested by observations such asdevelopmental anomalies in autistic patients, increased incidence ofautism in siblings of autistic patients, and a tendency for both of aset of monozygotic twins to be either autistic or not autistic (alsocalled “concordance” for a disorder). However, in most (75-80%) autisticindividuals, no underlying cause is found for the autism. Previousstudies have implicated abnormalities involving neurotransmittersincluding serotonin, norepineplrine, and histamine in some cases ofautism. Other causitive factors may include rubella, problems duringpregnancy, labor and delivery, cytomegalic inclusion disease,phenylketonuria, and Fragile X syndrome. Autistic children are also atincreased risk of developing seizure disorders, e.g., epilepsy,especially during their teen years.

A number of different treatments for autism have been developed. Many ofthe treatments, however, address the symptoms of the disease rather thanthe causes. For example, therapies ranging from psychoanalysis topsychopharmacology have been employed in the treatment of autism.Although some clinical symptoms may be lessened by these treatments,modest improvement, at best, has been demonstrated in only a minorfraction of the cases. Only a small percentage of autistic personsbecome able to function as self-sufficient adults.

Down's syndrome, a major cause of congenital mental retardation, is alsothe most common human birth defect. Down's syndrome occurs in about oneout of every 800 newborns, with the incidence increasing markedly in theoffspring of women over 35. Affecting an estimated one millionAmericans, it is the leading genetic cause of mental retardation and isassociated with a shorter than average life expectancy. Other symptomsare heart and intestinal defects, problems with the immune and endocrinesystems, and raft of tissue and skeletal deformities.

Over 90 percent of the individuals affected with Down's syndrome have anextra number 21 chromosome in all of their cells, giving each cell atotal of 47 chromosomes rather than the normal 46. For this reason, thecondition is also known as “Trisomy 21”. Trisomy 21 results fromnondisjunction or failure of chromosomes to separate sometime duringeither division of meiosis or mitosis. Most Down's syndrome individualshave trisomy 21, and conversely, in individuals who carry atranslocation involving chromosome 21, and in mosaics who have bothtrisomic and normal cells, the characteristics of the syndrome are seen.There are, however, rare forms of Down syndrome in which only part ofchromosome 21 is present in triplicate.

The present invention is further illustrated by the following examples,which are not intended to be limiting in any way.

EXEMPLIFICATION Example 1 Chemical Induction of mGluR5- and ProteinSynthesis-Dependent Long-Term Depression in Hippocampal Area CA1

Specific patterns of synaptic stimulation can induce long-termdepression (LTD) in area CA 10f the hippocampus. The LTD depends onactivation of metabotropic glutamate receptors (mGluRs) and rapidprotein synthesis. As described herein this form of synapticmodification can be induced by brief application of the selective mGluRagonist (RS)-3,5-dihydroxyphenylglycine (DHPG). DHPG-LTD is a saturableform of synaptic plasticity, requires mGluR5, is mechanisticallydistinct from N-methyl-D-aspartate receptor (NMDAR)-dependent LTD, andshares a common expression mechanism with protein synthesis-dependentLTD evoked using synaptic stimulation. DHPG-LTD can be useful forbiochemical analysis of mGluR5- and protein synthesis-dependent synapticmodification.

Introduction

Homosynaptic long-term depression (LTD) is a widely expressed form ofsynaptic plasticity in the brain. The best understood type of LTD isinduced in hippocampal area CA1 by low-frequency synaptic stimulation(LFS) via an N-methyl-D-aspartate (NMDA) receptor-dependent rise inpostsynaptic intracellular Ca²⁺ and the activation of a proteinphosphatase cascade (Bear and Abraham (1996)). Under the appropriatecircumstances, pharmacological activation of NMDA receptors (NMDARs) canalso induce this type of LTD. This “chem-LTD” approach has been usefulfor the biochemical characterization of the mechanism, revealing, forexample, that NMDAR-dependent LTD is associated with dephosphorylationof the GluR1 subunit of the postsynapticα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor(Lee et al. 1998).

Recent work has shown that mechanistically distinct types of LTD canalso be induced in CA1 by other types of synaptic stimulation. Forexample, paired-pulse stimulation repeated at 1 Hz for 15 min (PP-LFS)induces LTD that is independent of NMDARs and requires activation ofmetabotropic glutamate receptors (mGluRs). This mGluR-dependent form ofLTD is of particular interest because it also requires rapid translationof preexisting mRNA. A “chem-LTD” approach could be particularly usefulfor dissecting this novel mechanism. Indeed, reports from several groupsindicate that transient activation of Group I mGluRs with the selectiveagonist (RS)-3,5-dihydroxyphenylglycine (DHPG) can induce LTD (Camodecaet al. 1999; Fitzjohn et al. 1999; Palmer et al. 1997). However, it isclear that not all protocols are equivalent; for example, some areeffective only under conditions of low Mg²⁺ and are partially dependenton NMDARs (Palmer et al. 1997; Schnabel et al. 1999).

Here we characterize a chemical induction protocol that reliablyproduces protein synthesis-dependent LTD (Huber et al. 2000). We showthat mGluR5 is required for LTD induction and provide novel evidencethat this chemically induced LTD shares a common saturable expressionmechanism with LTD induced using PP-LFS. We anticipate that the methodwe describe here will be useful for understanding how mGluR activationregulates mRNA translation and the expression of synaptic LTD.

Methods

All animals were used in accordance with procedures approved by theBrown University Institutional Animal Care and Use Committee.Hippocampal slices were prepared from postnatal day 21-30 (P21-30) LongEvans rats (Charles River, Cambridge, Mass.) and mGluR5 knockout mice(Lu et al. 1997) as described previously (Huber et al. 2000). For mostexperiments, CA3 was removed immediately after sectioning. Slicesrecovered for 1-2 h at room temperature (rats) or at 30° C. (mice) inartificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 5KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 1 MgCl₂, 2 CaCl₂, and 10 dextrose,saturated with 95% O₂-5% CO₂. For recording, slices were placed in asubmersion recording chamber and perfused with 30° C. ACSF at a rate of2 m/min.

Synaptically evoked field potentials (FPs) were recorded from area CA1as described previously. Sharp microelectrode and whole cellvoltage-clamp recordings were made using Axoclamp 2B and Axopatch 1Damplifiers (Axon Instruments), respectively. Sharp electrodes (80-120MΩ) were filled with 3 M K-acetate and 10 mM KCl; patch pipettes (3-7MΩ) were filled with (in mM) 134 K-gluconate, 6 KCl, 4 NaCl, 10 HEPES,0.2 EGTA, 4 MgATP, 0.3 TrisGTP, and 14 phosphocreatine. The pH of theinternal solution was adjusted to 7.25 with KOH, and the osmolarity wasadjusted to 300 mOsm with H₂O or sucrose. Only experiments in whichthere was less than a 15% change in series resistance were included inthe analysis. Waveforms were filtered at 2 kHz and acquired anddigitized at 10 kHz on a PC using Experimenter's Workbench (DataWaveSystems, Boulder, Colo.).

Baseline responses were collected every 10-30 s using a stimulationintensity (10-30 μA; 0.2 ms) yielding 50-60% of the maximal response.Experiments in which there was a >5% drift in the response magnitudeduring the 20-min baseline period before DHPG or LFS were excluded fromfurther analysis. All experiments with mGluR5 KO mice used wildtypelittermates as controls and were performed blind to the genotype, laterdetermined by Therion (Troy, N.Y.). LFS consisted of 900 pulses at 1 Hz.PP-LFS consisted of 900 pairs of stimuli (50-ms interstimulus interval)delivered at I Hz. In saturation experiments, stimulus duration wasincreased from 0.2 to 0.4 ms during PP-LFS.

The group data were analyzed as follows: 1) the initial slopes of theFPs and excitatory postsynaptic potentials (EPSPs), or the amplitude ofthe excitatory postsynaptic currents (EPSCs), for each experiment wereexpressed as percentages of the preconditioning or DHPG baselineaverage, 2) the lime scale in each experiment as converted to time fromthe onset of conditioning or DHPG, and 3) the time-matched, normalizeddata were averaged across experiments and expressed in the text andfigures as the means±SE. Significant differences between groups weredetermined using an independent t-test or ANOVA performed on a 5-minaverage taken 1 h after LFS or DHPG application.

R,S-DHPG and D-2-amino-5-phosphonopentanoic acid (D-AP5) was purchasedfrom Tocris (St. Louis, Mo.); all other chemicals were from SigmaChemical (St Louis, Mo.). DHPG was prepared as a 100 times stock in H₂O,aliquoted and stored at −20° C. Fresh stocks were made once a week. A 10times stock of AP5 was prepared in ACSF and stored at 4° C. These stockswere diluted in ACSF to achieve their final concentrations. Picrotoxinwas dissolved directly into ACSF immediately before use.

Results

Application of DHPG for 5 min produced an acute, dose-dependentdepression of evoked FPs (FIG. 1A). At concentrations ≧50 μM, the FP didnot fully recover after drug wash out. Instead, the synaptic responsesstabilized at a depressed level (50 μM: 69±5%, means±SE, of pre-DHPGbaseline; n=11; 100 μM: 48±1%; n=4). In all subsequent studies 50 μM,DHPG (5 min) was used to induce what we will refer to as DHPG-LTD.Application of another Group I mGluR agonist, quisqualic acid (5 min; 5μM), also resulted in LTD (81±2%; n 4), confirming that the effect isnot peculiar to DHPG. Two-pathway experiments (n=4), in which only oneinput was stimulated during DHPG, indicated that DHPG-LTD does notrequire concurrent synaptic stimulation (stimulated: 62±4%;unstimulated: 68±5%, P>0.2; FIG. 1B). DHPG-LTD also showed evidence ofsaturation; two applications of 50 μM DHPG were sufficient to producemaximal depression (FIG. 1C).

Intracellular recordings confirmed that the DHPG-LTD of FPs reflectsdiminished synaptic transmission. Both sharp electrode recording ofEPSPs and whole cell voltage-clamp recording of EPSCs (recorded at −70mV) revealed stable LTD (EPSP: 61±5%; n=6; FIG. 1D; EPSC: 69±5%; n=5;FIG. 1E). In contrast, there were no significant long-term changes inmembrane potential, input resistance, or membrane excitability measured1 h after DHPG (data not shown). Thus DHPG-LTD is a long-lastingmodification of synaptic transmission.

The competitive NMDAR antagonist AP5 (50 μM) had no effect on themagnitude of DHPG-LTD as compared with interleaved control slices (AP5:83±3%, n=5; control: 85±3%, n=4; P>0.3; FIG. 2A). LTD induced with 5 μMquisqualic acid was also unaffected by AP5 (79±2%; n=3). Therefore LTDinduced by pharmacological activation of Group I mGluRs under theseexperimental conditions does not require concurrent NMDAR activation.

To assess the involvement of mGluR5, the major Group I mGluR in area CA1pyramidal neurons (Romano et al. 1995), DHPG-LTD was attempted in micelacking this receptor. DHPG-LTD was absent in the mGluR5 homozygousmutants (98±3% measured 1 h after DHPG application; n=8; FIG. 2A). Anintermediate amount of LTD was observed in heterozygous mutants (84±4%;n=6), as compared with wild-type littermate controls (77±2%; n=9; FIG.2B). A one-way ANOVA revealed a significant effect of genotype[F(2,19)=10.33, P<0.001]. A subsequent Tukey test revealed that both thewild-type and heterozygotes were significantly different fromhomozygotes (P<0.025). Although there is a trend for DHPG-LTD in theheterozygotes to be less than wildtypes, this is not significant(P=0.5). Thus DHPG-LTD strictly relies on mGluR5, and the presence ofone allele for mGluR5 is sufficient for LTD induction. In contrast toDHPG-LTD, normal NMDAR-dependent LTD, induced with LFS, was observed inthe homozygous mutants (87±2%; n=6; P>0.6; FIG. 2C) as compared with thewild type mice (89±5%; n=6). These results indicate that there are twodistinct routes of LTD induction in area CA1: one that relies on NMDARsand another on mGluR5.

The results from the mGluR5 knockouts indicate that the inductionmechanisms of NMDAR-dependent LTD and DHPG-LTD are different. The nextexperiment was designed to test whether these two forms of LTD utilizesimilar expression mechanisms. Repeated episodes of LFS were deliveredto saturate NMDAR-dependent LTD (FIG. 3A). DHPG then was then applied,and the magnitude of LTD was measured by renormalizing FP slope valuesto a pre-DHPG baseline. If NMDAR-dependent LTD and DHPG-LTD utilize acommon expression mechanism, then previous saturation of NMDAR-dependentLTD should reduce or occlude DHPG-LTD. However, DHPG still significantlydepressed synaptic responses (81±5% of pre-DHPG baseline; n=5; P<0.05;FIG. 3B), suggesting that NMDAR-dependent LTD and DHPG-LTD use distinctexpression mechanisms.

The same approach was used to assess whether DHPG-LTD employs the samesaturable expression mechanism as synaptically evoked mGluR-dependentLTD. PP-LFS in the presence of the NMDAR antagonist D-AP5 (50 μM) wasused to saturate mGluR-dependent LTD, and DHPG (50 μM) was then appliedto the slice (FIG. 3C). In contrast to the previous occlusionexperiment, DHPG application after saturation of LTD with PP-LFS did notinduce any further LTD (100±5% of pre-DHPG baseline; n=5; P>0.5; FIG.3D). These results provide strong evidence that mGluR-LTD induced withDHPG and PP-LFS share common expression mechanisms.

Discussion

A number of different protocols have been introduced to inducehomosynaptic LTD in CA1 (Berretta and Cherubini 1998; Camodeca et al.1999; Dudek and Bear 1992; Fitzjohn et al. 1999; Huber et al. 2000; Kempand Bashir 1999; Oliet et al. 1997; Overstreet et al. 1997; Palmer etal. 1997). Although mGluR involvement has been suggested for many ofthese, the constellation of findings is confusing and not entirelyconsistent with a single mGluR-dependent form of LTD. For example, ithas been reported that application of 100 μM DHPG for 10 min to adulthippocampal slices elicits little LTD unless slice excitability isincreased by removing Mg²⁺ from the extracellular medium (Palmer et al.1997; Schnabel et al. 1999). The resulting LTD is partially blocked byNMDAR antagonists. Moreover, PP-LFS in adult hippocampal slices canapparently elicit LTD via activation of either Group I mGluRs oractivation of AMPA/kainate receptors (Kemp and Bashir 1999). Incontrast, we recently demonstrated that in P21-30 rats, both PP-LFS andDHPG (50 μM, 5 min) induce LTD that is 1) independent of NMDARactivation, 2) blocked entirely by mGluR antagonists, and 3) dependenton a transient phase of mRNA translation (Huber et al. 2000). The latterfinding is of particular importance, as this mGluR-LTD model should beuseful for elucidating the regulation and function of dendritic proteinsynthesis, which may be defective in Fragile-X mental retardation (Jinand Warren 2000).

Because of the diverse effects of DHPG and PP-LFS, it could not beassumed that previous findings under different experimental conditionswould apply to our model. Therefore it was necessary to characterize theprotein synthesis-dependent form of mGluR-LTD. We have shown here thatDHPG-LTD is a saturable form of synaptic plasticity, that it requiresmGluR5, that it is mechanistically distinct from NMDAR-dependent LTD,and, importantly, that it shares a common saturable expression mechanismwith the LTD evoked using PP-LFS. Because DHPG-LTD does not requireconcurrent synaptic stimulation, it is a form of “chem-LTD” (Lee et al.1998) that should be useful for biochemical and biophysical studies.

REFERENCES

-   Bear M F, and Abraham W C. Long-term depression in hippocampus. Annu    Rev Neurosci 19: 437-462, 1996.-   Berretta N, and Cherubini E. A novel form of long-term depression in    the CA1 area of the adult rat hippocampus independent of glutamate    receptors activation. Eur J Neurosci 10: 2957-2963, 1998.-   Camodeca N, Breakwell N A, Rowan M J, and Anwyl R. Induction of LTD    by activation of Group I mGluR in dentate gyrus in vitro.    Neuropharmacology 38: 1597-1606, 1999.-   Dudek S M, and Bear M F. Homosynaptic long-term depression in area    CA1 of hippocampus and effects of N-methyl-D-aspartate receptor    blockade. Proc Natl Acad Sci USA 89: 4363-4367, 1992.-   Fitzjohn S M, Kingston A E, Lodge D, and Collingridge G L.    DHPG-induced LTD in area CA1 of juvenile rat hippocampus;    characterisation and sensitivity to novel mGlu antagonists.    Neuropharmacology 38: 1577-1584, 1999.-   Huber K M, Kayser M S, and Bear M F. Role for rapid dendritic    protein synthesis in hippocampal mGluR-dependent long-term    depression. Science 288: 1254-1257, 2000.-   Jin P, and Warren S T. Understanding the molecular basis of Fragile    X syndrome. Hum Mol Genet 9: 901-908, 2000.-   Kemp N, and Bashir Z I. Induction of LTD in the adult hippocampus by    the synaptic activation of AMPA/kainate and metabotropic glutamate    receptors. Neuropharmacology 38: 495-504, 1999.-   Lee H K, Kameyama K, Huganir R L, and Bear M F. NMDA induces    long-term synaptic depression and dephosphorylation of the GluR1    subunit of AMPA receptors in hippocampus. Neuron 21: 1151-1162,    1998.-   Lu Y M, Jia Z, Janus C, Henderson J T, Gerlai R, Wojtowicz J M, and    Roder J C. Mice lacking metabotropic glutamate receptor 5 show    impaired learning and reduced CA1 long-term potentiation (LTP) but    normal CA3 LTP. J Neuroscience 17: 5196-5205, 1997.-   Oliet S H, Malenka R C, and Nicoll R A. Two distinct forms of    long-term depression coexist in CA1 hippocampal pyramidal cells.    Neuron 18: 969-982, 1997.-   Overstreet L S, Pasternak J F, Colley Pa., Slater N T, and Trommer    B L. Metabotropic glutamate receptor mediated long-term depression    in developing hippocampus. Neuropharmacology 36: 831-844, 1997.-   Palmer M J, Irving A J, Seabrook G R, Jane Del., and Collingridge    G L. The group I mGlu receptor agonist DHPG induces a novel form of    LTD in the CA1 region of the hippocampus. Neuropharmacology 36:    1517-1532, 1997.-   Romano C, Sesma M A, McDonald Conn., O'Malley K, van der Pol A, and    Olney J W. Distribution of metabotropic glutamate receptor mGluR5    immunoreactivity in rat brain. J Comp Neurol 355: 455-469, 1995.-   Schnabel R, Kilpatrick I C, and Collingridge G L. An investigation    into signal transduction mechanisms involved in DHPG-induced LTD in    the CA1 region of the hippocampus. Neuropharmacology 38: 1585-1596,    1999.

EXAMPLE 2 Internalization of Ionotropic Glutamate Receptors in responseto mGluR activation

Activation of Group I mGluR I metabotropic glutamate receptors (mGluRs)stimulates dendritic protein synthesis and long-term synaptic depression(LTD), but it remains unclear how these effects are related. Here weprovide evidence that a consequence of mGluR activation in thehippocampus is the rapid loss of both AMPA and NMDA receptors fromsynapses. Like mGluR-LTD, the stable expression of this change requiresprotein synthesis. These data suggest that expression of mGluR-LTD is atleast partly postsynaptic, and that a functional consequence ofdendritic protein synthesis is the regulation of glutamate receptortrafficking.

Introduction

Two mechanistically distinct forms of homosynaptic long-term depression(LTD) coexist in the hippocampus. Induction of one form depends onactivation of N-methyl-D-aspartate receptors (NMDARs) and postsynapticprotein phosphatases, and induction of the other depends on activationof postsynaptic Group I metabotropic glutamate receptors (mGluRs) andthe local translation of dendritic mRNA¹. There is strong support forthe idea that NMDAR-dependent LTD (NMDA-LTD) is a consequence of reducedsynaptic expression of α-amino-3-hydroxy-5-methylisoxazole-4-propionatereceptors (AMPARs)²⁻⁷. Less is known about expression of mGluR-dependentLTD (mGluR-LTD), although a presynaptic mechanism has beensuggested^(8,9.)

Until recently, progress on mGluR-LTD has been hampered by the lack of areliable synaptic induction protocol. An alternative method has been totransiently activate Group I mGluRs with the selective agonist(R,S)-3,5-dihydroxyphenylglycine (DHPG)¹⁰⁻³. In hippocampal slices, DHPG(50 μM, 5 min) induces LTD in that requires protein synthesis¹³, andthat seems to use the same saturable expression mechanism as mGluR-LTDevoked with patterned synaptic activity¹⁴. Therefore, we used thischemical induction protocol on hippocampal neurons in culture and inslices to investigate the possibility that mGluR-LTD is expressed as achange in postsynaptic glutamate receptor expression.

Results

1) DHPG Stimulates Internalization of AMPARs

To examine the effect of mGluR activation on AMPARs expressed on thesurface of hippocampal neurons, we used an acid-strip immunocytochemicalstaining protocol³. Surface receptors on living cultured hippocampalneurons were labeled with antibodies directed against the extracellularN-terminus of the GluR1 subunit. The cells were treated with either DHPG(50 ΞM, 5 min) or control medium and, after various intervals, theremaining surface antibodies were stripped away with an acetic acidwash. The neurons were fixed, and immunocytochemistry was done undermembrane-permeabilizing conditions to detect antibodies bound tointernalized AMPARs. All analyses were performed blind, withoutexperimenter knowledge of the treatment conditions.

DHPG application for 5 minutes stimulated a greater than 2-fold increasein internalized GluR1 puncta that was observed as early as 15 minutesafter treatment onset (puncta per 10 μm of dendrite, control, 0.62±0.09,n=65 cells; DHPG, 1.44±0.17, n=60 cells; p<0.0002) and persisted for atleast 1 hour (control, 0.58±0.08, n=42 cells; DHPG, 1.14±0.15, n=38cells; FIGS. 4A and B). The increased internalization of GluR1 was aspecific consequence of activating Group I mGluRs, as it was completelyblocked by the mGluR antagonist LY344545 (ref. 15; 100 μM; control,0.42±0.10, n=15; DHPG, 1.39±0.34, n=14; LY344545 alone, 0.32±0.08, n=13;DHPG+LY344545, 0.29±0.04, n=17; FIG. 4C). In contrast, the NMDARantagonist 2-amino-5-phosphonovaleric acid (APV, 50 μM) had no effect(control, 0.74±0.19, n=7; DHPG, 1.49±0.22, n=10; DHPG+APV, 1.51±0.3,n=10).

Stable expression of mGluR-LTD requires dendritic protein synthesis. Wefound that pretreatment of cultures with the mRNA translation inhibitorcycloheximide (chx, 60 μM, applied 15 min before DHPG) alsosignificantly inhibited the DHPG-induced increase in internalized GluR1measured at 60 minutes (control, 0.85±0.14, n=24; DHPG, 1.5±0.27, n=20;DHPG+chx, 1.02±0.12, n=25, different from DHPG alone at p<0.03, FIG.4D). A mechanistically distinct protein synthesis inhibitor, anisomycin,also blocked mGluR-stimulated endocytosis (data not shown). Neithercycloheximide nor anisomycin had any significant effect on basal levelsof internalized puncta (control+chx, 0.76±0.09, n=10, FIG. 4D).

2) Surface AMPARs are Lost Following DHPG Treatment

We next determined if the DHPG-induced increase in internalized AMPARsis accompanied by a net decrease in surface-expressed receptor clustersat synapses. At various intervals after DHPG washout, cells were fixedand surface GluR2 or GluR1 was labeled with N-terminal antibodieswithout permeabilization. The cultures were then permeabilized, andsynapses were labeled using an antibody against the presynaptic markersynapsin I or synaptophysin coupled to the appropriate secondaryantibody. Under control conditions, most synapses were immunoreactivefor AMPAR clusters (GluR2, 80.6±9.0%; n=10 cells, 200 synapses, FIGS.5A-D; GluR1, 72.5±4.7%; n=15 cells, 225 synapses; FIGS. 5E and F).

The percentage of synapses with AMPAR clusters was dramatically reducedby DHPG treatment. Only 40.8±11% of synapses had surface staining forGluR2 (n=10 cells, 200 synapses; p<0.03) measured 1 hour after treatment(FIGS. 5G-I). Similar results were obtained in additional experimentswith GluR1 (29.3±5.4% GluR1-positive synapses 15 min after DHPGtreatment, n=14 cells, 210 synapses; 20.0±12.0% GluR1-positive synapses60 min after DHPG treatment, n=15 cells, 225 synapses; FIGS. 5J and K).

Pretreatment of cultures with cycloheximide (60 μM, applied 15 minbefore DHPG) inhibited the DHPG-induced decrease in synaptic GluR1clusters measured at 60 minutes (synapses with GluR1, 55.7±5.1%, n=15cells, 225 synapses; p<0.05 versus DHPG alone; FIG. 5K). However, thenumber of GluR1-positive synapses decreased 15 minutes after DHPG onsetin the presence of the inhibitor (synapses with GluR1, 37.8±3.8%, n=15cells, 225 synapses; FIG. 5J). These findings suggest that proteinsynthesis is involved in determining the fate of internalized receptors,but not in the initial endocytosis stimulated by mGluR activation.

To confirm the effect of mGluR activation on surface AMPARs using analternative approach, we treated high-density cultures with DHPG (50 μM,5 min) or control medium and surface receptors were labeled with biotin60 minutes later. Biotinylated receptors were precipitated and the ratioof surface to total GluR1 was determined by quantitative westernblotting. This biochemical analysis confirmed that surface AMPARs arereduced by DHPG treatment to only 56.8±4.0% of the value in controlcultures (n=4 in each treatment group; p<0.01; FIG. 6).

3) DHPG application reduces mEPSC frequency

The immunocytochemical and biochemical experiments suggest that DHPGtreatment is likely to have a significant effect on AMPAR-mediatedsynaptic transmission in cultured neurons. To investigate thispossibility directly, we examined the effect of DHPG on AMPAR-mediatedmEPSCs. As reported for other manipulations that stimulate receptorinternalization (for example, see ref. 16), we observed a significantdecrease in the frequency of mEPSCs. The inter-event interval was 315%of baseline at 15 min after DHPG treatment (n=11 cells, p<0.05) and 319%of baseline at 60 minutes (n=9 cells, p<0.002; FIG. 7).

In addition to the change in frequency, there was also a trend towardattenuated mEPSC amplitude at 15 (94.2% baseline; n=11 cells) and 60(92.2% baseline; n=9 cells; FIG. 7) minutes following DHPG, but thiseffect did not achieve statistical significance. Considered togetherwith the imaging and biochemical results, the most straightforwardinterpretation of the mEPSC data is that DHPG silences a discretepopulation of synapses because its entire complement of AMPARs isinternalized.

4) Surface NMDARs are Lost Following DHPG Treatment

NMDAR activation has been reported to stimulate a loss of synapticAMPARs without affecting NMDARs². To determine if mGluR-stimulationaffects NMDAR clusters, cells were treated with DHPG, fixed and stainedwith an N-terminal antibody for the NR1 subunit of the NMDAR undernon-permeabilizing conditions. The cells were then permeabilized andsynapses were labeled using an antibody against synapsin I. In controlneurons, 67±4% of synapses (n=20 cells, 300 synapses) contained NR1immunoreactive puncta (FIGS. 8A-D and G). Following DHPG treatment, thepercentage of NR1-positive synapses was reduced to 28±6% at 15 minutes(n=16 cells, 240 synapses, p<0.003) and 21±3% at 60 minutes (n=19 cells,285 synapses; FIGS. 8E and G). As was the case for AMPARs, the change insurface NR1 clusters following DHPG was significantly attenuated at 60minutes when the cultures were treated with cycloheximide (42±5%NR1-labeled at 60 min; n=20 cells, 300 synapses; p<0.05 versus DHPGalone; FIG. 8G).

The loss of NMDARs from synapses following DHPG was surprising. To ruleout the possibility of nonspecific changes in the postsynaptic neurons,we monitored changes in the distribution of synaptic GABA_(A) receptorsusing an antibody against the N-terminal of the β₁, subunit. Unlikesynapses with glutamate receptors, DHPG had no effect on the percentageof synapsin-labeled puncta with GABA_(A)β₁ clusters (control, 11.8±4%,n=10 cells, 150 synapses; 60 min after DHPG treatment, 10.9±2%, n=10;data not shown). To corroborate the loss of surface NMDARs followingDHPG, high-density cultures were treated with DHPG (50 μM, 5 min, n=5),DHPG+cycloheximide (60 μM; n=4), or control medium (n=5), and surfaceNMDARs were labeled with biotin 60 minutes later. Biotinylated receptorswere precipitated and the ratio of surface to total NR1 was determinedby quantitative western blotting (FIGS. 8H and I). This analysisconfirmed that surface NMDARs are significantly reduced by DHPGtreatment to 32.3±8.2% of the value in control cultures, and that thischange is inhibited by cycloheximide (79.1±14.5% of control; FIG. 8I).

5) LTD of NMDAR-EPSCs

The loss of synaptic NR1 clusters clearly distinguishes the effect ofDHPG from other treatments that selectively affect AMPARs^(2,17-19).Thus, our data suggest that in addition to the depression ofAMPAR-mediated synaptic transmission, induction of mGluR-LTD should alsoaffect transmission mediated by NMDARs. To test this hypothesis, wechemically induced LTD in hippocampal slices from postnatal day 21-28(P21-28) rats with DHPG¹⁴ as we monitored NMDAR mediated excitatorypostsynaptic currents (EPSCs) in CA1 neurons voltage clamped at +40 mV,as described previously²⁰. These experiments revealed that applicationof DHPG (5 min) produced a dose-dependent LTD of NMDAR-EPSCs (EPSCamplitude 30 minutes after DHPG treatment as percent of baseline, 50 μM,70.7±2.9, n=3, p<0.05; 100 μM: 57.7±1.0, n=5; different from baseline atp<0.00005, paired 1-test; FIG. 9A.

As an additional test for an mGluR-induced loss of NMDAR function, weexamined the effects of 1100 μM DHPG (5 min) on currents evoked by NMDAapplied near the proximal portion of the primary apical dendrite (FIG.9B). Significant depression of NMDAR currents occurred (percent baselineat 50-60 min after DHPG treatment, DHPG, 61.1±12.0, n=7; control,97.3±9.4; n=7;p<0.05); however, the time course of this change was muchslower than that observed for synaptically evoked EPSCs. Unlike theEPSCs, which depressed immediately, the NMDA-evoked currents transientlypotentiated (as described previously with the agonist1-amino-cyclopentane-1,3 dicarboxylic acid (ACPD) ^(10, 21)) and thenslowly decreased over the course of an hour. The early LTD of EPSCscould be accounted for by a presynaptic mechanism or by the rapiddispersal of synaptic NMDARs (without immediate internalization).Migration of NMDARs within the membrane has been demonstrated both incultured cells²² and in slices²³. Regardless of the early consequences,however, the parallel depression of NMDAR EPSCs and NMDA-evokedresponses 60 minutes after DHPG treatment is consistent with an eventualreduction in surface NMDAR expression during mGluR-LTD.

Discussion

Our data demonstrate that activation of Group I mGluRs in culturedhippocampal neurons stimulates internalization of synaptic AMPA and NMDAreceptors, and that the stable expression of these changes is sensitiveto protein synthesis inhibitors. The same DHPG treatment (50 μM, 5 min)in hippocampal slices stimulates mGluR-LTD that depends uponpostsynaptic mRNA translation¹³ and, as we now show, is expressed as achange in NMDAR— as well as AMPAR-mediated transmission. Thus, removalof synaptic glutamate receptors is a candidate mechanism for theexpression of mGluR-LTD in the hippocampus. This notion is consistentwith the finding that cerebellar LTD, which is also triggered byactivation of Group I mGluRs, requires postsynaptic endocytosis ofAMPARs²⁴.

Hippocampal mGluR-LTD was previously shown to be associated with areduced frequency of spontaneous and evoked postsynaptic responseswhich, according to the traditional assumptions of quantal analysis,suggested a presynaptic expression mechanism^(8,9). However, these dataare also consistent with ‘synaptic silencing,’ arising from the completeloss of receptors at an activated synapse^(16,17,25)

Similar to what we observe following mGluR activation, NMDA-LTD isassociated with a reduced expression of postsynaptic AMPARs (and adecreased frequency of spontaneous excitatory postsynaptic currents²).In principle, the two routes of LTD induction could converge on a commonsaturable expression mechanism at the same synapses; however, thishypothesis is at odds with the finding that mGluR-LTD and NMDA-LTD arenot mutually occluding^(9,14). An alternative is that mGluRs and NMDARsregulate separate populations of AMPARs, perhaps at distinct populationsof synapses.

Several previous studies suggested that synaptic NMDARs are relativelystatic in comparison to AMPARs^(4,19,27). However, we find that bothNMDARs and AMPARs are internalized with a similar time-course (<15 min)following DHPG treatment. Rapid endocytosis of NMDARs has also beendemonstrated in immature cortical cultures under basal conditions²⁸.This receptor internalization was inhibited by the binding of thepostsynaptic density protein PSD95 to the C-terminus of the NR2Bsubunit. Thus, a potential mechanism for DHPG-stimulated NMDARendocytosis could involve regulation of the interaction of PSD95 andNR2B.

Besides their obvious relevance to hippocampal mGluR-LTD, we suggest ourfindings may be of additional significance. First, we show a unique rolefor protein synthesis that, considered with previous findings^(13,26,29)is likely to occur in the postsynaptic neuron as a specific consequenceof synaptic activity. Using glutamate receptor trafficking as an assay,this preparation should be very useful for dissecting the molecularmechanisms that couple mGluR activation to dendritic mRNA translationregulation. Second, the loss of ionotropic receptors on hippocampalneurons following DHPG is reminiscent of what happens at theneuromuscular junction before synapse elimination³⁰, and Group I mGluRshave recently been implicated in the loss of climbing fiber synapses inthe developing cerebellum³¹. Thus, the model we describe here should beuseful for testing the long-standing hypothesis that mGluRs and themechanisms of LTD are involved in activity-dependent synapse eliminationin the cerebral cortex^(32,33).

Methods

1) Acid Strip Immunocytochemical Protocol

Low-density cultures of rat hippocampal neurons were made as previouslydescribed³⁴. All rats were housed in the Brown University Animal CareFacility and all procedures were approved by Brown University AnimalCare and Use Committee. Briefly, the hippocampus was removed from E18rat fetuses, trypsinized (0.25%), dissociated by trituration, and platedonto poly-L-lysine (1 mg/ml) coated glass coverslips (80,000 cells/ml)for 4 h. The coverslips were then transferred to dishes containing amonolayer of glial cells in growth medium and the neurons were allowedto mature for 14-22 days. Surface AMPARs were labeled on live cells withan antibody directed against the extracellular N-terminus of the GluR1subunit (amino acids 271-285; 5 μg per ml; Oncogene Research, San Diego,Calif., and a gift of R. Huganir). The neurons were then treated with aspecific agonist of the Group I mGluR5 DHPG, 50 μM in medium) or controlmedium for 5 min. Ten or fifty-five minutes following treatment, thecells were chilled in 4° C. Tris-buffered saline (TBS) to stopendocytosis, and then exposed to 0.5 M NaCl/0.2 M acetic acid (pH 3.5)for 4 min on ice to remove antibody bound to extracellular GluR1.Cultures were rinsed and fixed in 4% paraformaldehyde with 4% sucrose.Nonspecific staining was blocked and cells were permeabilized in TBScontaining 0.1% Triton-X, 4% goat serum and 2% BSA. Internalized primaryantibody was made visible by incubation with a Cy3-labeled secondaryantibody for 1 h (1: 300). In the initial studies, treatments included 1μM tetrodotoxin and 1 μM ω-conotoxin to limit depolarization-inducedneurotransmitter release. We later found that identical results wereobtained without ω-conotoxin, so this treatment was subsequentlyomitted.

2) Immunocytochemical Localization of Synaptic Receptors

Following experimental treatment, low-density cultures were fixed in 4%paraformaldehyde with 4% sucrose for 5 min. Cultures were rinsed in PBSand then blocked in PBS with 20% fetal bovine serum for 1 h. Cultureswere stained with N-terminal receptor antibodies overnight at 4° C.(GluR2, 1: 100, Chemicon, Ternecula, Calif.; GluR1, 1: 100, gift of R.Huganir; NR1, 1: 500, Chemicon MAB363; GABA_(A)β₁, 1: 100 Santa CruzBiologicals, Santa Cruz, Calif.). Cultures were then rinsed in blockingbuffer containing 0.1% Triton-X for 20 min and exposed to antibodiesdirected against presynaptic proteins (synapsin 1, 1: 1000, Chemicon;synaptophysin, 1: 100, Boehringer Manheim, Irvine, Calif.) for 1 h atroom temperature. Cultures were then rinsed and exposed to theappropriate fluorescent secondary antibodies (Jackson Immunoresearch,West Grove, Pa.).

3) Analysis of Immunocytochemical Data

Microscopy was performed with a Nikon E800 microscope using a 60×1.4 NAobjective (Melville, N.Y.). Fluorescence images were collected with aSensys cooled CCD camera and analyzed using IP-Labs software. Additionalimages were collected with a Olympus Flowview confocal microsope with a60×1.2 NA objective. All analyses were performed blind to thestimulation history of the culture. Microscopic fields had 1-3 neuronsdisplaying smooth soma and generally healthy morphology with multipledistinct processes. Immunofluorescence was analyzed along the proximal50 μm of 3 or more dendrites per neuron. Immunoreactive puncta weredefined as discrete points along the dendrite with fluorescenceintensity twice the background staining of the neuron. Five cells wereanalyzed per culture and 3-6 cultures were analyzed per condition.Separate controls were performed with each experiment and a Student'st-test was used to determine statistical significance. Data areexpressed as puncta per 10 μm of dendrite unless stated otherwise.

4) Biochemical Measurements of Surface Expressed Receptors

Biotinylation experiments were performed as previously described³⁵.Briefly, 2-week-old high-density cultured hippocampal neurons weretreated with either control medium or 50 μM DHPG for 5 min, andincubated for 1 h at 37° C. to allow endocytosis to occur. The sistercultures were placed on ice to stop endocytosis and washed two timeswith ice-cold artificial cerebrospinal fluid (ACSF) containing 124 mMNaCl, 5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 0.8 mM MgCl₂, 1.8 mMCaCl₂, 10 mM dextrose, and saturated with 95% O₂, 5% CO₂. Cultures werethen incubated with ACSF containing 1 mg/ml Sulfo-NHS-LC-Biotin (PierceChemical Company, Rockford, Ill.) for 30 min on ice. Cultures wererinsed in TBS to quench the biotin reaction. Cultures were lysed in 300μl of modified RIPA buffer (1% Triton X-100, 0.1% SDS, 0.5% deoxycholicacid, 50 mM NaPO₄, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 10 mM sodiumpyrophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and 1 mg/mlleupeptin). The homogenates were centrifuged at 14,000×g for 15 min at4° C. Fifteen microliters (5%) of the supernatant were removed tomeasure total GluR1 or NR1; 200 μl (66.67%) of the remaining supernatantwas incubated with 100 μl of 50% Neutravidin agarose (Pierce ChemicalCompany) for 3 h at 4° C., washed 3 times with RIPA buffer, and boundproteins were resuspended in 40 μl of SDS sample buffer and boiled.Quantitative western blots were performed on both total and biotinylated(surface) proteins using anti-GluR1 C-terminal (1: 1000, UpstateBioteclmology, Lake Placid, N.Y.) and anti-NR1 N-terminal antibodies (1:1000, Chemicon). Immunoreactive bands were visualized by enhancedchemiluminescence (ECL, Amersham, Piscataway, N.J.) captured onautoradiography film (Amersham Hyperfilm ECL). Digital images, producedby densitometric scans of autoradiographs on a ScanJet IIcx (HewlettPackard, Palo Alto, Calif.) with DeskScan II software (Hewlett Packard),were quantified using NIH Image 1.60 software. The surface/total ratiowas calculated for each culture, and treatment groups were comparedusing a paired t-test. Control experiments confirmed that theintracellular protein actin was not biotinylated in this assay. Fordisplay purposes, the data are expressed as the ratio of DHPG to controlvalues.

5) mEPSC Recordings and Analysis

Cultured hippocampal cells at room temperature were superfused at 1ml/min in medium consisting of 140 mM NaCl, 3.5 mM KCl, 10 mM HEPES, 20mM glucose, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 0.05 mM picrotoxin, 0.001 mMTTX, and pH was adjusted to 7.4 with NaOH. Patch electrodes (4-5 mΩ)were filled with 116 mM Kgluconate, 6 mM KCl, 20 mM HEPES, 0.5 mM EGTA,2 mM NaCl, 4 mM Mg-ATP, 0.3 mM Na-GTP, 10 mM sodium phosphocreatine,adjusted to pH 7.3 and osmolarity ˜300 mM. Cells were voltage-clamped at−60 mV (near the resting membrane potential of the cells), and mEPSCswere amplified using the Axopatch 1D amplifier. Recordings were filteredat 2 kHz, digitized at 10 kHz, and stored on a computer usingExperimenter's Workbench (DataWave Systems, Boulder, Colo.) and onvideotape. Series and input resistances were monitored throughout theexperiment and only those cells stable (<15% change) in these parameterswere included in the analysis. Average input resistance was ˜600 MΩ andaverage series resistance was ˜15 MΩ. Events were detected off-lineusing an automatic detection program (MiniAnalysis, Synaptosoft,Decatur, Ga.) with a detection threshold set at a value greater than atleast two standard deviations of the noise values. The detectionthreshold remained constant for the duration of each experiment. Onlyevents with a monotonic rise time and exponential decay were included inthe analysis. Inter-event interval and mEPSC amplitude were comparedduring a 10-min baseline period and in 10-min windows 15 and 60 minutesafter 50 μM DHPG application for 5 min. Due to non-normal distributionsof mEPSC parameters, statistics were performed using the Wilcoxonsigned-ranks test and significance was placed at p<0.05.

6) Hippocampal Slice Physiology

Hippocampal slices were prepared from P21-30 Long Evans rats (CharlesRiver, Cambridge, Mass.) as described previously^(13,14). Slicesrecovered for 1-2 -h at room temperature in artificial cerebrospinalfluid (ACSF) containing 124 mM NaCl, 5 mM KCl, 1.25 mM NaH₂PO₄, 26 mMNaHCO₃, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM dextrose, saturated with 95% O₂,5% CO₂. For recording, slices were placed in a submersion recordingchamber and perfused with 30° C. ACSF at a rate of 2 ml/min.

Synaptically evoked NMDAR-mediated EPSCs were recorded from area CA1 asdescribed previously for visual cortex²⁰. NMDA-evoked currents wereexamined by picospritzing 1 mM NMDA (made in ACSF), applied for 3.5-12.5ms, near the proximal portion of the primary apical dendrite.NMDA-evoked currents were elicited once every two minutes. Stimulationintensity or picospritz pulse duration/pressure were adjusted to evokean inward current with amplitude of 50 pA or greater.

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EXAMPLE 3 Fragile X Mental Retardation

Fragile X syndrome is a prevalent form of inherited mental retardation,occurring with a frequency of 1 in 4000 males and 1 in 8000 females. Thesyndrome is also characterized by developmental delay, hyperactivity,attention deficit disorder, and autistic-like behaviors (Jin, P., etal., Hum Mol Genet, 9: 901-8, (2000)). There is no effective treatmentfor Fragile X syndrome.

The syndrome is typically caused by a repeat expansion mutation in theFMR1 (Fragile X Mental Retardation 1) gene that encodes FMRP (Fragile XMental Retardation Protein). FMRP associates with translatingpolyribosomes and a subset of brain mRNAs, and functions as a regulatorof protein synthesis (Feng, Y., et al., Mol Cell 1: 109-18 (1997);Brown, V., et al., Cell 107: 477-87 (2001); Darnell, J. C., et al., Cell107: 489-99 (2001); Zhang, Y. Q., et al., Cell 107: 591-603 (2001)).Polyribosomes, FMR1 mRNA, and FMRP are present in dendritic spines, themajor site of synaptic transmission on cortical neurons (Weiler, I. J.,et al., Am J Med Genet 83: 248-52 (1999)). The Fmr1 null mutant (Fmr1-KO(knock out)) mouse, which has a behavioral phenotype consistent withFragile X syndrome, was employed to determine the involvement of FMRP insynaptic plasticity. Protein synthesis-dependent, late-phase long-termsynaptic potentiation (LTP) is unaffected in the hippocampus of mutantmice (Godfraind, J. M., et al., Am J Med Genet 64: 246-251 (1996);Paradee, W., et al., Neuroscience 94: 185-92 (1999)).

Local, synaptic control of protein synthesis is required for stableexpression of a second form of hippocampal synaptic modification:long-term synaptic depression (LTD). As shown herein, LTD is triggeredby activation of Group I metabotropic glutamate receptors (mGluRs). FMRPmay be involved in this form of synaptic plasticity since FMRP is one ofthe proteins synthesized in response to mGluR activation (Weiler, I. J.,et al., Am J Med Genet 83: 248-52(1999)).

As shown herein, mGluR-dependent LTD (mGluR-LTD), a proteinsynthesis-dependent form of synaptic plasticity, is significantlyactivated in the hippocampus of Fmr1-KO mice. FMRP normally functions asa negative regulator of translation (Zhang, Y. Q., et al., Cell 107:591-603 (2001); Laggerbauer, B., et al., Hum Mol Genet 10: 329-38(2001); Li, Z., et al., Nucleic Acids Res 29: 2276-83 (2001)). FMRP hasan important functional role in regulating activity-dependent synapticplasticity. Thus, exaggerated LTD and/or mGluR function may beresponsible for aspects of the behavioral phenotype in Fragile Xsyndrome, and antagonists of Group I mGluRs (e.g., mGluR5, mGluR1) canbe employed as therapeutic agents in the treatment of Fragile Xsyndrome.

Materials and Methods

Hippocampal slices were prepared from postnatal day (P) 21-30, C57BL/6congenic Fmr1-KO mice and their wildtype littermates as previouslydescribed. Briefly, slices were collected in ice-cold dissection buffercontaining 212 mM sucrose; 2.6 mM KCl; 1.25 mM NaH2PO₄; 26 mM NaHCO₃; 5mM MgCl₂; 0.5 mM CaCl₂; and 10 mM dextrose. The CA3 position of thehippocampus was removed immediately after sectioning. Slices recoveredwere placed for 1-5 hr at 30° C. in artificial cerebrospinal fluid(ACSF) containing 124 mM NaCl; 5 mM KCl; 1.25 mM NaH₂PO₄; 26 mM NaHCO₃;1 mM MgCl₂; 2 mM CaCl₂; 10 mM dextrose, saturated with 95% O₂, 5% CO₂.For recording of field potentials, slices were placed in a submersionrecording chamber and perfused with 30° C. ACSF at a rate of 2 ml/min.

Field potentials (FPs) were recorded with extracellular recordingelectrodes filled with ACSF and placed in the stratum radiatum of areaCA1. Synaptic responses were evoked by a 200 μsec current pulse toSchaffer collateral axons with a concentric bipolar tungsten stimulatingelectrode. Stable baseline responses were collected every 30 sec using astimulation intensity (10-30 μA) yielding 50-60% of the maximalresponse. mGluR-LTD was induced in the presence of theN-methyl-D-aspartate (NMDA) receptor (NMDAR) antagonistD-(−)-2-amino-5-phosphono-pentanoic acid (D-APV) (50 μM) usingpaired-pulse low frequency stimulation (PP-LFS) consisting of 900 pairsof stimuli (50 msec interstimulus interval) delivered at 1 Hz. NMDAR-LTDwas induced using 900 single pulses delivered at 1 Hz (Dudek, S. M., etal., Proc Natl Acad Sci USA 89: 4363-7 (1992)).

Waveforms were filtered at 2 kHz, acquired and digitized at 10 kHz on aPC using Experimenter's Workbench (DataWave Systems, Boulder, Colo.).The group data were analyzed as follows: (1) the initial slope of the FPfor each experiment was expressed as percentages of the pre-conditioningor 3,5-dehydroxyphenylglycine (DHPG) baseline average, and (2) the timescale in each experiment was converted to time from the onset ofpre-conditioning or DHPG baseline average. All experiments wereperformed blind to the genotype of the mice, determined after analysisof individual experiments. After genotyping, the time-matched,normalized data were averaged across experiments and expressed in thetext and figures as the means (±SEM). Significant differences betweengroups were determined using an independent t-test and Komolgarov-Smimovtest.

R,S-DHPG ((R,S)-3,5-dihydroxyphenylglycine) and APV were purchased fromTocris (St. Louis, Mo.). All other chemicals were purchased from SigmaChemical Co. (St Louis, Mo.). DHPG was prepared as a 100× stock in H₂O,aliquoted and stored at −20° C. Fresh stocks were made once a week. A10× stock of APV was prepared in ACSF and stored at 4° C. The stockswere diluted in ACSF to achieve their final concentrations.

Results

Normal Synaptic Transmission in Fmr1-KO Mice

Hippocampal slices were prepared from postnatal day 21-30, C57BL/6congenic Fmr1-KO mice and their wildtype littermates. Excitatorysynaptic field potentials evoked by stimulation of the Schaffercollaterals were recorded extracellularly from the stratum radiatum ofarea CA1. In all cases, the experimenters were blind to the genotype.

Previous studies have examined the properties of transmission atSchaffer collateral synapses in the CA1 region of hippocampus of thesemutant mice. In terms of basal transmission, excitability, paired-pulsefacilitation, early phase LTP elicited with 100 Hz stimulation, andlate-phase (protein synthesis-dependent) LTP induced with theta-burststimulation, Fmr1-KO mice were indistinguishable from wild-type (WT)littermates (Godfraind, J. M., et al., Am J Med Genet 64: 246-51 (1996);Paradee, W., et al., Neuroscience 94: 185-92 (1999)). Thus, theexcitatory synaptic transmission mediated by AMPA and NMDA receptors,and the state of inhibition, are not appreciably affected by the absenceof FMRP. Field potential amplitudes, in response to increasing stimuluscurrent, were not different between Fmr1-KO and wildtype littermates(F(1,350)=0.358, P>0.5), the maximum amplitude of field potentials fromFmr1-KO mice (1.73±0.19 mV; n=39 slices from 17 mice) was not differentfrom wildtype (WT; 1.63±0.16 mV; n=36 slices from 18 mice; p=0.36), andthe stimulus currents used to evoke baseline responses were notdifferent between groups (Fmr1-KO 22±1 mA; WT 23±2 mA).

mGluR-LTD Induced by Synaptic Stimulation is Enhanced in Fmr1-KO Mice

As shown herein, paired-pulse stimulation repeated at 1 Hz for 15minutes (PP-LFS) induces LTD that is independent of NMDA receptors, andrequires activation of Group I mGluRs and the rapid translation ofpreexisting mRNA. The consequences of PP-LFS hippocampal in slices fromKO (knock out) and WT (wild type) animals was also determined herein.

PP-LFS (delivered in the presence of 50 μM D-2-amino-5-phosphonovalerate(APV) to block NMDA receptors) produced a significant LTD in WT mice(93±3% 60 min following PP-LFS; n=21 slices from 10 mice; FIGS. 10A, 10Band 10C).

The magnitude of LTD in these experiments is considerably less thanpreviously reported in rats as described above, which may be due to thespecies and strain of mice employed. The magnitude of LTD induced withPP-LFS was significantly increased in slices prepared from KO animals(82±3%; n=18 slices from 8 mice; different from WT at p<0.004; t-test).The difference was initially observed about 15 minutes after the tetanusand there was no indication that responses during or immediately afterthe PP-LFS were different in KO and WT animals (FIGS. 10A and 10B). FIG.10A depicts the average time course of the change in FPs followingPP-LFS. LTD in KO animals measured 82±3% of pre-PP-LFS baseline (n=18slices from 8 mice; open circles) as compared to 93±2% in WT controls(n=21 slices from 10 mice; filled circles; different at p=0.004,t-test). FIG. 10B depicts representative FPs (2 min average) taken atthe times indicated by the numbers on the graph. Scale bars: 1 mV, 5msec (1,2) and 1 mV, 10 msec (PP-LFS).

The Kolmolgarov-5 miniov test on the cumulative probabilitydistribution, showed that the distribution of depression values wasdifferent between KO and WT groups was significantly different (p<0.05;FIG. 10C). FIG. 10C depicts cumulative probability distributions of FPslope values (% of baseline), measured on hour after PP-LFS inindividual slices from both KO and WT groups.

mGluR-LTD induced by DHPG is enhanced in Fmr1-KO mice mGluR LTD can beinduced by the selective Group I mGluR agonist DHPG((RS)-3,5-dihydroxyphenylglycine) which more uniformly affects synapsesand circumvents the need for presynaptic activation. Dose-dependentinduction of LTD following DHPG (50-100 μM, 5 min) occurs. As shownherein, activation of mGluR5 is required for induction, and proteinsynthesis is required for stable expression, of DHPG-LTD. LTD withPP-LFS and DHPG are mutually occluding, which may indicate they utilizethe same saturable expression mechanism. DHPG was employed to induceplasticity to determine whether mGluR-LTD is increased in Fmr1-KO miceusing an independent method.

As described above, experiments were performed in the presence of APV toeliminate the confound of NMDA receptor-dependent synapticmodifications. DHPG (100 μM, 5 min) induced a saturating level of LTD.The data show a significant enhancement of mGluR-LTD in slices from KOmice (FIGS. 11A, 11B and 11C).

DHPG application to slices from Fmr1-KO mice resulted in depression ofFP slope values to 77±3% of pre-DHPG baseline (measured 60 min afterDHPG application n=21 slices from 9 mice). In comparison, DHPG-inducedLTD was 88±4% in WT mice (15 slices from 8 animals, p=0.02; FIGS. 11A,11B). FIG. 11A depicts the average (±SEM) field potential (FP) slopevalues over the time course of the experiment. In Fmr1-KO animals, theresponse 60 min after treatment was depressed to 77±3% of pre-DHPGbaseline (n=21 slices from 9 mice; open circles); in interleaved WTcontrols, the response was depressed to 88±4% of baseline (n=15 slicesfrom 8 mice; filled circles; different at p=0.02; t-test). FIG. 11Bdepicts representative FPs (2 min average) taken at the times indicatedby the numbers on the graph (Scale bar: 1 mV; 5 msec).

The Kolmolgarov-Smirnov test performed on the cumulative probabilitydistribution confirmed the statistical significance of this difference(p<0.05; FIG. 11C). FIG. 11C depicts cumulative probabilitydistributions of FP slope values (% of baseline), measured one hourafter DHPG in individual hippocampal slices from both KO and WT groups.

Although the acute effect of DHPG on synaptic transmission also appearedto be slightly enhanced in Fmr1-KO slices, this difference was notstatistically significant (maximal acute depression: WT: 36±4%, KO:26±5% of pre-DHPG baseline values). Western blots of hippocampalhomogenates also confirmed that mGluR5 levels are comparable in KO andWT mice.

NMDA Receptor-Dependent LTD is Normal in Fmr1-KO Mice

Two forms of homosynaptic LTD coexist at CA3-CA1 synapses: mGluR-LTD anda form which is triggered by activation of NMDA receptors (NMDAR-LTD)(Oliet, S. H. et al., Neuron 18: 969-82 (1997)). As shown herein,NMDAR-LTD in hippocampal slices is independent of mGluR activation andprotein synthesis, and requires activation of postsynaptic proteinphosphatases. To determine whether FMRP selectively regulates proteinsynthesis-dependent plasticity or LTD mechanisms in general NMDAR-LTD inFmr1-KO mice was examined. NMDAR-LTD was elicited by delivering 900single pulses at 1 Hz (Dudek, S. M., et al., Proc Natl Acad Sci USA 89:4363-7 (1992)). In contrast to mGluR-LTD, NMDAR-LTD was normal inFmr1-KO mice (86±4%; 14 slices from 8 animals) as compared to WTlittermates (84±4%; 12 slices from 4 animals; p=0.6; FIGS. 12A, 12B and12C).

FIG. 12A depicts the average time course of the change in FPs followinglow-frequency stimulation (LFS). LTD in KO animals measured 86±4% ofpre-LFS baseline (n=14 slices from 8 mice; open circles) as compared to84±4% in wild-type controls (n=12 slices from 4 mice; filled circles;p=0.6, t-test). FIG. 12B depicts the representative FPs (2 min average)taken at the times indicated by the numbers on the graph (Scale bar: 1mV, 10 msec). FIG. 12C depicts the cumulative probability distributionsof FP slope values (% of baseline), measured on hour after LFS inindividual slices from both KO and WT groups.

The data described herein show that FMRP specifically regulates mGluR—and protein synthesis-dependent synaptic plasticity. As shown herein,using two distinct induction protocols, mGluR-dependent LTD issignificantly increased in the hippocampus of animals lacking FMRP. FMRPregulates LTD downstream of the mGluRs, likely at the level of rRNAtranslation. Since Fragile X syndrome is related to exaggeratedmGluR-dependent synaptic plasticity, drugs that inhibit Group I mGluRsand/or LTD can be used for the treatment of neurological disordersincluding Fragile X syndrome.

Discussion

Involvement of FMRP in the Regulation of LTD

Activation of postsynaptic Group I mGluRs (e.g., mGluR5 and mGluR1), bythe selective agonist (e.g., DHPG) or by synaptically releasedglutamate, triggers LTD at Schaffer collateral synapses in area CA1 ofthe hippocampus. One expression mechanism for the LTD of synaptictransmission is the internalization of AMPA and NMDA receptors (Snyder,E. M., et al., Nat Neurosci 4: 1079-85 (2001); Xiao, M. Y., et al.,Neuropharmacology 41: 664-71 (2001)). Both synaptic depression andglutamate receptor internalization initiated by in GluR activationwithout new protein synthesis, but the stable expression of the changefails to occur when mRNA translation (but not transcription) isinhibited (see above). The critical site of protein synthesis is thepostsynaptic dendrite.

An mRNA that is translated in response to postsynaptic Group I mGluRactivation encodes FMRP (Weiler, I. J., et al., Am J Med Genet 83:248-52 (1999)). Thus, the data described herein shows that themGluR-dependent synthesis of FMRP plays a role in the stabilization ofLTD. FMRP can function as a negative regulator of mRNA translation(Zhang, Y. Q., et al., Cell 107: 591-603 (2001); Laggerbauer, B., etal., Hum Mol Genet 10: 329-38 (2001); Li, Z., et al., Nucleic Acids Res29: 2276-83 (2001)). FMRP normally serves to limit expression of LTD byinhibiting mGluR-dependent translation of other synaptic mRNAs (FIG.13).

As shown in FIG. 13, activation of mGluR5 stimulates the internalizationof AMPA receptors (AMPAR) and NMDA receptors. The stable expression ofthis modification requires protein synthesis, which may be negativelyregulated by FMRP synthesized in response to mGluR activation.Therefore, in the absence of FMRP, LTD magnitude is increased.

An mRNA that is negatively regulated by FMRP encodes the microtubuleassociated protein MAP1b, which has been shown in Drosophila to regulatesynaptic structure and function (Zhang, Y. Q., et al., Cell 107: 591-603(2001)). An increase of MAP 1b mRNA on polyribosomes in cells derivedfrom patients with Fragile X syndrome, has recently been reported.

In addition to LTD triggered by activation of Group I mGluRs,homosynaptic LTD can be is induced by activating NMDA receptors (Bear,M. F., et al., Annu Rev Neurosci 19: 437-62 (1996)). In hippocampalslices, expression of NMDAR-mediated LTD is not protein synthesisdependent for at least 1 hr and does not occlude mGluR-mediated LTD.Normal NMDAR-LTD in the Fmr1-KO mice shows that these forms of LTDutilize distinct mechanisms. Another form of NMDAR-dependent plasticity,LTP, is also unaffected in Fmr1 knockout mice (Godfraind, J. M., et al.,Am J Med Genet 4: 246-51 (1996); Paradee, W., et al., Neuroscience 94:185-92 (1999)). The data described herein show that FMRP may beselectively involved in synaptic modifications that are triggered inresponse to mGluR-stimulated protein synthesis.

Role of LTD and FMRP in Cortical Development

LTD and LTP may normally work in concert to fine-tune patterns ofsynaptic connectivity during development (Bear, M. F., et al., Science237: 42-8 (1987); Bear, M. F., et al. J. Neurobiol 41: 83-91 (1999) andto store memories in the adult brain (Bear, M. F., Proc Natl Acad SciUSA 93: 13453-9 (1996)). As shown herein, activation of mGluRs incultured hippocampal neurons is a long-term decrease in the surfaceexpression of the ionotropic glutamate receptors that mediate synaptictransmission, possibly as a prelude to synapse elimination (Snyder, E.M., et al., Nat Neurosci 4: 1079-85 (2001)). Thus, in the absence ofFMRP, enhanced LTD may interfere with the establishment and maintenanceof strong synapses required for normal brain function.

Dendritic spine development is slowed in the cerebral cortex of Fmr1-KOmice (Nimchinsky, E. A., et at., J Neurosci 21: 5139-46 (2001)).Dendritic spines are the major targets of glutamatergic synapses in thecortex. Synapses are formed during development when long, thinprotospines emitted by pyramidal cell dendrites make contact with nearbyaxons (Dailey, M. E., et al., J Neurosci 16: 2983-94 (1996)). As thesynapse stabilizes, the spines shorten and become fatter. An increasedpercentage of long, thin dendritic processes, reminiscent ofprotospines, is a characteristic feature of cortical neurons inFMRP-deficient mice (Nimchinsky, E. A., et al., J Neurosci 21: 5139-46(2001); Comery, T. A., et al., Proc Natl Acad Sci USA 94: 5401-4 (1997))and affected humans (Hinton, V. J., et al., Am J Med Genet 41: 289-94(1991); Irwin, S. A., et al., Am J Med Genet 98: 161-7 (2001)). Anunderlying defect may enhance activity- and mGluR-dependent synapseturnover, abnormally prolonging a state in which neurons are activelyseeking new synaptic input. Hippocampal neurons in culture expresssignificantly longer, thinner spines following DHPG treatment, whichrequires protein synthesis (Vanderklish, P. W., et al., Proc Natl AcadSci USA 99: 1639-44 (2002)).

Treatment of Fragile X Syndrome

The association of Group I mGluRs and activity-dependent proteinsynthesis is not restricted to early postnatal development, the cerebralcortex, or LTD. mGluR— and protein synthesis-dependent LTD can beelicited in hippocampus from mature animals, where it may contribute tomemory storage (Zheng, H., et al., Cell 107: 617-29 (2001)),particularly during novel or stressful situations (Bear, M. F., ProcNatl Acad Sci USA 6: 9457-8 (1999); Braunewell, K. H., et al., RevNeurosci 12: 121-40 (2001)). LTD in the cerebellum, which depends onGroup I mGluRs, may contribute to learning motor reflexes (Bear, M. F.,et al., In “Synapses,” eds. Cowan, W. M., Sudhoff, et al., The JohnsHopkins University Press, Baltimore, pp. 455-517 (2001)), requires rapidtranslation of mRNA (Karachot, L., et al., J Neurophysiol 86: 280-9(2001)). mGluR-triggered protein synthesis in the hippocampus can reducethe threshold for synaptic potentiation (Raymond, C. R., et al., J.Neurosci 20: 969-76 (2000)) and trigger epileptiform activity (Merlin,L. R., et al., J Neurophysiol 80: 989-93 (1998); Wong, R. K., et al.,Adv Neurol 79: 685-98 (1999)). FMRP may normally function as a negativefeedback regulator of these physiological processes. The prominentfeatures of Fragile X syndrome also include heightened responses tonovelty, compulsions, and seizures.

The methods described herein can be used to treat these heightenedresponses to novelty, compulsions, seizures, anxiety and epilepsy in ahuman with a neurological disorder or condition, in particular a humanwith Fragile X syndrome.

Fragile X mental retardation may be a consequence of increasedmGluR-dependent protein synthesis and/or LTD in the brain, both duringearly postnatal development and in adulthood. LTD magnitude increaseswith increasing activation of mGluR5. Titration of a competitiveantagonist may produce a graded reduction in mGluR— and proteinsynthesis-dependent response. These data show that Group I mGlu5 can beuseful to treat neurological disorders including Fragile X syndrome.

All patents, publications, and other references cited above are herebyincorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating anxiety in a human comprising the step ofadministering an effective amount of to Group I mGluR antagonist to ahuman having anxiety associated with fragile X syndrome.
 2. The methodof claim 1, wherein the Group I mGluR antagonist is selected from thegroup consisting of (E)-6-methyl-2-styryl-pyridine (SIB 1893),6-methyl-2-(phenylazo)-3-pyridinol, α-methyl-4-carboxyphenylglycine(MCPG), and 2-methyl-6-(phenylethynyl)-pyridine (MPEP).
 3. The method ofclaim 1 wherein the Group I mGluR antagonist is a mGluR5 antagonist. 4.The method of claim 1, wherein the Group I mGluR antagonist is a mGluR1antagonist.
 5. The method of Claim 1, wherein the Group I mGluRantagonist binds to a Group I mGluR.
 6. The method of claim 1, whereinthe Group I mGluR antagonist inhibits mGluR interaction with a G proteininvolved in a Group I mGluR receptor signal transduction.
 7. The methodof claim 1, wherein the Group I mGluR antagonist is administered in adose ranging from about 10 to about 1000 mg/kg body weight/day.
 8. Amethod of treating an epilepsy in a human comprising the step ofadministering an effective amount of Group I mGluR antagonist to a humanhaving epilepsy associated with fragile X syndrome.
 9. The method ofclaim 8, wherein the Group I mGluR antagonist is selected from the groupconsisting of (E)-6-methyl-2-styryl-pyridine (SIB 1893),6-methyl-2-(phenylazo)-3-pyridinol, α-methyl-4-carboxyphenylglycine(MCPG), and 2-methyl-6-(phenylethynyl)-pyridine (MPEP).
 10. The methodof claim 8, wherein the epilepsy in the human being treated with theGroup I mGluR antagonist is a benign childhood epilepsy.
 11. The methodof claim 8, wherein the Group I mGluR antagonist is a mGluR5 antagonist.12. The method of claim 8, wherein the Group I mOluR antagonist is amGluR1 antagonist.
 13. The method of claim 8, wherein the Group I mGluRantagonist binds to a Group I mGluR.
 14. The method of claim 8, whereinthe Group I mGluR antagonist inhibits mGluR interaction with a G proteininvolved in a Group I mGluR receptor signal transduction.
 15. The methodof claim 8, wherein the Group I mGluR antagonist is administered in adose ranging from about 10 to about 1000 mg/kg body weight/day.
 16. Amethod of treating anxiety in a human, comprising the step ofadministering an effective amount of a mGluR1 antagonist to a humanhaving anxiety associated with fragile X syndrome.
 17. A method oftreating anxiety in a human, comprising the step of administering aneffective amount of a mGluR5 antagonist to a human having anxietyassociated with fragile X syndrome.
 18. A method of treating epilepsy ina human, comprising the step of administering an effective amount of amGluR1 antagonist to a human having epilepsy associated with fragile Xsyndrome.
 19. A method of treating epilepsy in a human, comprising thestep of administering an effective amount of mGluR5 antagonist to ahuman having epilepsy associated with fragile X syndrome.